Plants having altered agronomic characteristics under nitrogen limiting conditions and related constructs and methods involving low nitrogen tolerance genes

ABSTRACT

Isolated polynucleotides and polypeptides, and recombinant DNA constructs useful for conferring improved nitrogen use efficiency and/or drought stress tolerance; compositions (such as plants or seeds) comprising these recombinant DNA constructs; and methods utilizing these recombinant DNA constructs are disclosed. The recombinant DNA constructs comprise a polynucleotide operably linked to a promoter that is functional in a plant, wherein said polynucleotides encode abiotic stress tolerance polypeptides.

FIELD

The field of the disclosure relates to plant breeding and genetics and,in particular, relates to recombinant DNA constructs useful in plantsfor conferring nitrogen use efficiency and/or tolerance to nitrogenlimiting conditions and/or drought conditions.

BACKGROUND

Stresses to plants may be caused by both biotic and abiotic agents. Forexample, biotic causes of stress include infection with pathogen, insectfeeding, and parasitism by another plant such as mistletoe. Abioticstresses include, for example, excessive or insufficient availablewater, nitrogen, temperature extremes, and synthetic chemicals such asherbicides.

Abiotic stresses such as drought, high salinity and deficiency ofnutrient elements adversely affect the growth and productivity of plantsincluding crops, which significantly limit crop production worldwide.Cumulatively, these factors are estimated to be responsible for anaverage 70% reduction in agricultural production. Plants are sessile andhave to adjust to the prevailing environmental conditions of theirsurroundings. This has led to their development of a great plasticity ingene regulation, morphogenesis, and metabolism. Adaptation and defensestrategies involve the activation of genes encoding proteins importantin the acclimation or defense towards the different stressors.

The absorption of nitrogen by plants plays an important role in theirgrowth (Gallais et al., J. Exp. Bot. 55(396):295-306 (2004)). Plantssynthesize amino acids from inorganic nitrogen in the environment.Consequently, nitrogen fertilization has been a powerful tool forincreasing the yield of cultivated plants, such as rice, maize andsoybean. Lack of sufficient plant-available nitrogen for optimum growthand development may be considered as an abiotic stress. In order toavoid pollution by nitrates and to maintain a sufficient profit margin,today farmers desire to reduce the use of nitrogen fertilizer. If aplant variety has increased nitrogen assimilation capacity, it wouldalso be expected to have increased growth and yield. In summary, plantvarieties that have better nitrogen use efficiency (NUE) are desirable.

Activation tagging can be utilized to identify genes with the ability toaffect a trait. This approach has been used in the model plant speciesArabidopsis thaliana (Weigel et al., Plant Physiol. 122:1003-1013(2000)). Insertions of transcriptional enhancer elements can dominantlyactivate and/or elevate the expression of nearby endogenous genes. Thismethod can be used to identify genes of interest for a particular trait(e.g. nitrogen use efficiency in a plant), that when placed in anorganism as a transgene, can alter that trait.

SUMMARY

The following embodiments are among those encompassed by the disclosure:

One embodiment, includes an isolated polynucleotide enhancing nitrogenstress tolerance of plant through over-expression, comprising: (a) apolynucleotide with nucleotide sequence of at least 85% sequenceidentity, based on the Clustal V method of alignment, to SEQ ID NO: 4,7, 10 or 13; (b) a polynucleotide with nucleotide sequence of at least85% sequence identity, based on the Clustal V method of alignment, toSEQ ID NO: 5, 8, 11 or 14; (c) a polynucleotide encoding a polypeptidewith amino acid sequence of at least 90% sequence identity, based on theClustal V method of alignment, to SEQ ID NO: 6, 9, 12 or 15; or (d) thefull complement of the nucleotide sequence of (a), (b) or (c), whereinover-expression of the polynucleotide in a plant enhances nitrogenstress tolerance. The nucleotide sequence comprises SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11, SEQ IDNO: 13 or SEQ ID NO: 14. The amino acid sequence of the polypeptidecomprises SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12 or SEQ ID NO: 15.

Another embodiment, includes a recombinant DNA construct comprising theisolated polynucleotide operably linked to at least one regulatorysequence, wherein the polynucleotide comprises (a) a polynucleotide withnucleotide sequence of at least 85% sequence identity, based on theClustal V method of alignment, to SEQ ID NO: 4, 5, 7, 8, 10, 11, 13 or14; (b) a polynucleotide encoding a polypeptide with amino acid sequenceof at least 90% sequence identity, based on the Clustal V method ofalignment, to SEQ ID NO: 6, 9, 12 or 15; or (c) the full complement ofthe nucleotide sequence of (a) or (b); the at least one regulatorysequence is a promoter functional in a plant.

A third embodiment, includes a plant or seed comprising a recombinantDNA construct comprising the polynucleotide operably linked to at leastone regulatory sequence, wherein the polynucleotide comprises (a) apolynucleotide with nucleotide sequence of at least 85% sequenceidentity, based on the Clustal V method of alignment, to SEQ ID NO: 4,5, 7, 8, 10, 11, 13 or 14; (b) a polynucleotide encoding a polypeptidewith amino acid sequence of at least 90% sequence identity, based on theClustal V method of alignment, to SEQ ID NO: 6, 9, 12 or 15; or (c) thefull complement of the nucleotide sequence of (a) or (b); the at leastone regulatory sequence is a promoter functional in a plant.

Another embodiment, includes a plant comprising in its genome arecombinant DNA construct comprising a polynucleotide operably linked toat least one regulatory element, wherein the polynucleotide comprises(a) a polynucleotide with nucleotide sequence of at least 85% sequenceidentity, based on the Clustal V method of alignment, to SEQ ID NO: 4,5, 7, 8, 10, 11, 13 or 14; (b) a polynucleotide encoding a polypeptidewith amino acid sequence of at least 90% sequence identity, based on theClustal V method of alignment, to SEQ ID NO: 6, 9, 12 or 15; or (c) thefull complement of the nucleotide sequence of (a) or (b); the said plantexhibits improved nitrogen use efficiency (NUE) when compared to acontrol plant.

Another embodiment, includes an isolated polynucleotide enhancingdrought tolerance of plant through over-expression, comprising: (a) apolynucleotide with nucleotide sequence of at least 85% sequenceidentity, based on the Clustal V method of alignment, to SEQ ID NO: 10;(b) a polynucleotide with nucleotide sequence of at least 85% sequenceidentity, based on the Clustal V method of alignment, to SEQ ID NO: 11;(c) a polynucleotide encoding a polypeptide with amino acid sequence ofat least 90% sequence identity, based on the Clustal V method ofalignment, to SEQ ID NO: 12; or (d) the full complement of thenucleotide sequence of (a), (b) or (c), wherein over-expression of thepolynucleotide in a plant enhances drought stress tolerance. Thenucleotide sequence comprises SEQ ID NO: 10 or SEQ ID NO: 11. The aminoacid sequence of the polypeptide comprises SEQ ID NO: 12.

Another embodiment, includes a recombinant DNA construct comprising theisolated polynucleotide operably linked to at least one regulatorysequence, wherein the polynucleotide comprises (a) a polynucleotide withnucleotide sequence of at least 85% sequence identity, based on theClustal V method of alignment, to SEQ ID NO: 10 or 11; (b) apolynucleotide encoding a polypeptide with amino acid sequence of atleast 90% sequence identity, based on the Clustal V method of alignment,to SEQ ID NO: 12; or (c) the full complement of the nucleotide sequenceof (a) or (b); the at least one regulatory sequence is a promoterfunctional in a plant.

Another embodiment, includes a plant or seed comprising a recombinantDNA construct comprising the polynucleotide operably linked to at leastone regulatory sequence, wherein the polynucleotide comprises (a) apolynucleotide with nucleotide sequence of at least 85% sequenceidentity, based on the Clustal V method of alignment, to SEQ ID NO: 10or 11; (b) a polynucleotide encoding a polypeptide with amino acidsequence of at least 90% sequence identity, based on the Clustal Vmethod of alignment, to SEQ ID NO: 12; or (c) the full complement of thenucleotide sequence of (a) or (b); the at least one regulatory sequenceis a promoter functional in a plant.

Another embodiment, includes a plant comprising in its genome arecombinant DNA construct comprising a polynucleotide operably linked toat least one regulatory element, wherein the polynucleotide comprises(a) a polynucleotide with nucleotide sequence of at least 85% sequenceidentity, based on the Clustal V method of alignment, to SEQ ID NO: 10or 11; (b) a polynucleotide encoding a polypeptide with amino acidsequence of at least 90% sequence identity, based on the Clustal Vmethod of alignment, to SEQ ID NO: 12; or (c) the full complement of thenucleotide sequence of (a) or (b); the said plant exhibits improveddrought when compared to a control plant.

A further embodiment, includes any of the plants of the disclosure,wherein the plant is selected from the group consisting of rice, maize,soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, barley,millet, sugar cane and switchgrass.

In another embodiment, a method of increasing nitrogen stress toleranceor NUE in a plant, comprising: (a) introducing into a regenerable plantcell a recombinant DNA construct comprising a polynucleotide operablylinked to at least one regulatory sequence, wherein the polynucleotideencodes a polypeptide having an amino acid sequence of at least 50%sequence identity, based on the Clustal V method of alignment, whencompared to SEQ ID NO: 6, 9, 12 or 15; (b) regenerating a transgenicplant from the regenerable plant cell after step (a), wherein thetransgenic plant comprises in its genome the recombinant DNA construct;and (c) obtaining a progeny plant derived from the transgenic plant ofstep (b), wherein said progeny plant comprises in its genome therecombinant DNA construct and exhibits increased nitrogen stresstolerance or NUE when compared to a control plant not comprising therecombinant DNA construct.

In another embodiment, a method of evaluating nitrogen stress toleranceor NUE in a plant, comprising: (a) introducing into a regenerable plantcell a recombinant DNA construct comprising a polynucleotide operablylinked to at least one regulatory sequence, wherein the polynucleotideencodes a polypeptide having an amino acid sequence of at least 50%sequence identity, based on the Clustal V method of alignment, whencompared to SEQ ID NO: 6, 9, 12 or 15; (b) regenerating a transgenicplant from the regenerable plant cell after step (a), wherein thetransgenic plant comprises in its genome the recombinant DNA construct;(c) obtaining a progeny plant derived from the transgenic plant, whereinthe progeny plant comprises in its genome the recombinant DNA construct;and (d) evaluating the progeny plant for nitrogen stress tolerance orNUE compared to a control plant not comprising the recombinant DNAconstruct.

In another embodiment, a method of determining an alteration of anagronomic characteristic in a plant, comprising: (a) introducing into aregenerable plant cell a recombinant DNA construct comprising apolynucleotide operably linked to at least one regulatory sequence,wherein the polynucleotide encodes a polypeptide having an amino acidsequence of at least 50% sequence identity, based on the Clustal Vmethod of alignment, when compared to SEQ ID NO: 6, 9, 12 or 15; (b)regenerating a transgenic plant from the regenerable plant cell afterstep (a), wherein the transgenic plant comprises in its genome therecombinant DNA construct; (c) obtaining a progeny plant derived fromthe transgenic plant, wherein the progeny plant comprises in its genomethe recombinant DNA construct; and (d) determining whether the progenyplant exhibits an alteration of at least one agronomic characteristicwhen compared to a control plant not comprising the recombinant DNAconstruct, wherein said determining step (d) comprises determiningwhether the transgenic plant exhibits an alteration of at least oneagronomic characteristic when compared, under nitrogen limitingconditions, to a control plant not comprising the recombinant DNAconstruct.

In another embodiment, a method of increasing drought stress tolerancein a plant, comprising: (a) introducing into a regenerable plant cell arecombinant DNA construct comprising a polynucleotide operably linked toat least one regulatory sequence, wherein the polynucleotide encodes apolypeptide having an amino acid sequence of at least 50% sequenceidentity, based on the Clustal V method of alignment, when compared toSEQ ID NO: 12; (b) regenerating a transgenic plant from the regenerableplant cell after step (a), wherein the transgenic plant comprises in itsgenome the recombinant DNA construct; and (c) obtaining a progeny plantderived from the transgenic plant of step (b), wherein said progenyplant comprises in its genome the recombinant DNA construct and exhibitsincreased drought stress tolerance when compared to a control plant notcomprising the recombinant DNA construct.

In another embodiment, a method of evaluating drought stress tolerancein a plant, comprising: (a) introducing into a regenerable plant cell arecombinant DNA construct comprising a polynucleotide operably linked toat least one regulatory sequence, wherein the polynucleotide encodes apolypeptide having an amino acid sequence of at least 50% sequenceidentity, based on the Clustal V method of alignment, when compared toSEQ ID NO: 12; (b) regenerating a transgenic plant from the regenerableplant cell after step (a), wherein the transgenic plant comprises in itsgenome the recombinant DNA construct; (c) obtaining a progeny plantderived from the transgenic plant, wherein the progeny plant comprisesin its genome the recombinant DNA construct; and (d) evaluating theprogeny plant for drought stress tolerance compared to a control plantnot comprising the recombinant DNA construct.

In another embodiment, the present disclosure concerns a recombinant DNAconstruct comprising any of the isolated polynucleotides of the presentdisclosure operably linked to at least one regulatory sequence, and acell, a plant, and a seed comprising the recombinant DNA construct. Thecell may be eukaryotic, e.g., a yeast, insect or plant cell, orprokaryotic, e.g., a bacterium.

BRIEF DESCRIPTION OF THE FIGURES

The disclosure can be more fully understood from the following detaileddescription of figure which forms a part of this application.

FIG. 1 shows the relative expression levels of OsLRP1 gene in leaves ofdifferent transgenic rice lines by real-time PCR analyses. The baseexpression level in DP0044.29 is set at 1.00, the numbers on the top ofthe columns are fold-changes compared to DP0044.29 rice. DP0005 is emptyvector transformed ZH11 rice plants.

FIG. 2 shows the relative expression levels of OsDN-LTP1 gene in leavesof different transgenic rice lines by real-time PCR analyses. The baseexpression level in ZH11-TC is set at 1.00, the numbers on the top ofthe columns are fold-changes compared to ZH11-TC rice.

FIG. 3 shows changes of soil volumetric water content at differentdevelopmental stage for drought testing OsDN-LNP1 transgenic rice. TheOsDN-LNP1 transgenic rice started heading at 37 days after stoppingwatering.

DESCRIPTION OF TABLES WITHIN THE SPECIFICATION

Table 1. SEQ ID NOs for nucleotide and amino acid sequences provided inthe sequence listing

Table 2. Rice gene names, Gene IDs (from TIGR) and Construct IDs

Table 3. Primers for cloning rice abiotic stress tolerance genes

Table 4. PCR reaction mixture for cloning abiotic stress tolerance gene

Table 5. PCR cycle conditions for abiotic stress tolerance gene

Table 6: Modified Hoagland's nutrient solution for culturing rice

Table 7. Low nitrogen assay of OsLRP1 transgenic rice plants undergreenhouse low nitrogen conditions (1^(st) experiment)

Table 8. Low nitrogen assay of OsLRP1 transgenic rice plants undergreenhouse low nitrogen conditions (2^(nd) experiment, ZH11-TC ascontrol)

Table 9. Low nitrogen assay of OsLRP1 transgenic rice plants undergreenhouse low nitrogen conditions (2^(nd) experiment, DP0158 ascontrol)

Table 10. Low nitrogen assay of OsDN-LTP1 transgenic rice plants undergreenhouse low nitrogen conditions (1^(st) experiment)

Table 11. Low nitrogen assay of OsDN-LTP1 transgenic rice plants undergreenhouse low nitrogen conditions (2^(nd) experiment, ZH11-TC ascontrol)

Table 12. Low nitrogen assay of OsDN-LTP1 transgenic rice plants undergreenhouse low nitrogen conditions (2^(nd) experiment, DP0158 ascontrol)

Table 13. Low nitrogen assay of OsDN-LTP1 transgenic rice plants undergreenhouse low nitrogen conditions (3^(rd) experiment, ZH11-TC ascontrol)

Table 14. Low nitrogen assay of OsDN-LTP1 transgenic rice plants undergreenhouse low nitrogen conditions (2^(nd) experiment, DP0158 ascontrol)

Table 15. Chlorate sensitive assay of OsDN-PPR1 transgenic riceseedlings at transgenic line level (1^(st) experiment)

Table 16. Chlorate sensitive assay of OsDN-PPR1 transgenic riceseedlings at transgenic line level (2^(nd) experiment)

Table 17. Chlorate sensitive assay of OsDN-LNP1 transgenic riceseedlings at transgenic line level (1^(st) experiment)

Table 18. Chlorate sensitive assay of OsDN-LNP1 transgenic riceseedlings at transgenic line level (2^(nd) experiment)

Table 19. Chlorate sensitive assay of OsRRM1 rice seedlings attransgenic line level (1^(st) experiment)

Table 20. Chlorate sensitive assay of OsRRM1 rice seedlings attransgenic line level (2^(nd) experiment)

Table 21. Grain yield analysis of OsLRP1 transgenic rice under field lownitrogen condition

Table 22. Grain yield analysis of OsLRP1 transgenic rice under fieldnormal nitrogen condition

Table 23. Biomass analysis of OsLRP1 transgenic rice under low nitrogencondition

Table 24. Plant height analysis of OsLRP1 transgenic rice under lownitrogen condition

Table 25. Plant height analysis of OsLRP1 transgenic rice under normalnitrogen condition

Table 26. Grain yield analysis of OsRRM1 transgenic rice under field lownitrogen condition

Table 27. Grain yield analysis of OsRRM1 transgenic rice under fieldnormal nitrogen condition

Table 28. Flag leaf SPAD value analysis of OsRRM1 transgenic rice underfield low nitrogen condition

Table 29. Top second leaf SPAD value analysis of OsRRM1 transgenic riceunder field low nitrogen condition

Table 30. Paraquat tolerance assay of OsDN-LNP1 transgenic rice plantsat transgenic line level (1^(st) experiment)

Table 31. Paraquat tolerance assay of OsDN-LNP1 transgenic rice plantsat transgenic line level (2^(nd) experiment)

Table 32. Grain yield analysis of OsDN-LNP1 transgenic rice plants underfield drought conditions

Table 33. Modified Hoagland's nutrient solution for culturingArabidopsis

Table 34. P values for green leaf area and greenness evaluated for theover-expression OsDN-PPR1 on 4 consecutive days under low nitrogencondition

Table 35. P values for green leaf area and greenness evaluated for theover expression OsRRM1 on 4 consecutive days under low nitrogencondition

Sequence Identification

TABLE 1 SEQ ID NOs for nucleotide and amino acid sequences provided inthe sequence listing Source SEQ ID NO: SEQ ID NO: species CloneDesignation (Nucleotide) (Amino Acid) Artificial DP0005 vector 1 n/aArtificial pBC-Yellow 2 n/a Artificial DsRED expression cassette 3 n/aOryza sativa OsDN-PPR1 4, 5 6 Oryza sativa OsLRP1 7, 8 9 Oryza sativaOsDN-LTP1 10, 11 12 Oryza sativa OsRRM1 13, 14 15 Artificial Primers16-27 n/a

The sequence descriptions and Sequence Listing attached hereto complywith the rules governing nucleotide and/or amino acid sequencedisclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825. The Sequence Listing contains the one letter code fornucleotide sequence characters and the three letter codes for aminoacids as defined in conformity with the IUPAC-IUBMB standards describedin Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219(2):345-373 (1984) which are herein incorporated by reference. Thesymbols and format used for nucleotide and amino acid sequence datacomply with the rules set forth in 37 C.F.R. § 1.822.

SEQ ID NO: 1 is the nucleotide sequence of vector DP0005.

SEQ ID NO: 2 is the nucleotide sequence of the pBC-yellow vector.

SEQ ID NO: 3 is the nucleotide sequence of DsRed expression cassette.

SEQ ID NO: 4 is the nucleotide sequence of cDNA of OsDN-PPR1.

SEQ ID NO: 5 is the nucleotide sequence of CDS of OsDN-PPR1.

SEQ ID NO: 6 is the amino acid sequence of OsDN-PPR1.

SEQ ID NO: 7 is the nucleotide sequence of cDNA of OsLRP1.

SEQ ID NO: 8 is the nucleotide sequence of CDS of OsLRP1.

SEQ ID NO: 9 is the amino acid sequence of OsLRP1.

SEQ ID NO: 10 is the nucleotide sequence of gDNA of OsDN-LTP1.

SEQ ID NO: 11 is the nucleotide sequence of CDS of OsDN-LTP1.

SEQ ID NO: 12 is the amino acid sequence of OsDN-LTP1.

SEQ ID NO: 13 the nucleotide sequence of cDNA of OsRRM1.

SEQ ID NO: 14 the nucleotide sequence of CDS of OsRRM1.

SEQ ID NO: 15 is the amino acid sequence of OsRRM1.

SEQ ID NO: 16 is forward primer for cloning cDNA of OsDN-PPR1.

SEQ ID NO: 17 is reverse primer for cloning cDNA of OsDN-PPR1.

SEQ ID NO: 18 is forward primer for cloning cDNA of OsLRP1.

SEQ ID NO: 19 is reverse primer for cloning cDNA of OsLRP1.

SEQ ID NO: 20 is forward primer for cloning gDNA of OsDN-LNP1.

SEQ ID NO: 21 is reverse primer for cloning gDNA of OsDN-LNP1.

SEQ ID NO: 22 is forward primer for cloning cDNA of OsRRM1.

SEQ ID NO: 23 is reverse primer for cloning cDNA of OsRRM1.

SEQ ID NO: 24 is forward primer for real-time RT-PCR analysis of OsLRP1gene.

SEQ ID NO: 25 is reverse primer for real-time RT-PCR analysis of OsLRP1gene

SEQ ID NO: 26 is forward primer for real-time RT-PCR analysis ofOsDN-LTP1 gene.

SEQ ID NO: 27 is reverse primer for real-time RT-PCR analysis ofOsDN-LTP1 gene.

DETAILED DESCRIPTION

The disclosure of each reference set forth herein is hereby incorporatedby reference in its entirety.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to “a plant” includes aplurality of such plants, reference to “a cell” includes one or morecells and equivalents thereof known to those skilled in the art, and soforth.

As used herein:

The term “OsDN-PPR1 (Pentatricopeptide Repeat)” refers to a ricepolypeptide that confers a low nitrogen tolerance phenotype and isencoded by the rice gene locus LOC_Os11g10740.1 “DN-PPR1 polypeptide”refers herein to the OsDN-PPR1 polypeptide and its homologs from otherorganisms.

The OsDN-PPR1 polypeptide (SEQ ID NO: 6) is encoded by the codingsequence (CDS) (SEQ ID NO: 5) or nucleotide sequence (SEQ ID NO: 4) atrice gene locus LOC_Os11g10740.1. This polypeptide which has fourpentatricopeptide repeat (PPR repeat) domains, is annotated as“tetratricopeptide-like helical, putative” in TIGR (the internet atrice.plantbiology.msu.edu/index.shtml), and “tetratricopeptide-likehelical domain containing protein/pentatricopeptide, putative” in NCBI(on the world web at ncbi.nlm.nih.gov/), however does not have any priorassigned function.

The term “OsLRP1 (Leucine Rich Repeat)” refers to a rice polypeptidethat confers a low nitrogen tolerance phenotype and is encoded by therice gene locus LOC_Os11g10720.1. “LRP1 polypeptide” refers herein tothe OsLRP1 polypeptide and its homologs from other organisms.

The OsLRP1 polypeptide (SEQ ID NO: 9) is encoded by the coding sequence(CDS) (SEQ ID NO: 8) or nucleotide sequence (SEQ ID NO: 7) at rice genelocus LOC_Os11g10720.1. This polypeptide is annotated as “Cf2/Cf5disease resistance protein, putative” in TIGR and “Leucine Rich Repeatfamily protein” in NCBI, however does not have any prior assignedfunction.

The term “OsDN-LTP1 (Low nitrogen Tolerance Protein)” refers to a ricepolypeptide that confers a low nitrogen tolerance phenotype. “DN-LTP1polypeptide” refers herein to the OsDN-LTP1 polypeptide and its homologsfrom other organisms. The OsDN-LTP1 polypeptide (SEQ ID NO: 12) isencoded by the coding sequence (CDS) (SEQ ID NO: 11) or nucleotidesequence (SEQ ID NO: 10)

The term “OsRRM1 (RNA Recognition Motif)” refers to a rice polypeptidethat confers a low nitrogen tolerance phenotype and is encoded by therice gene locus LOC_Os06g50890.1. “RRM1 polypeptide” refers herein tothe OsRRM1 polypeptide and its homologs from other organisms.

The OsRRM1 polypeptide (SEQ ID NO: 15) is encoded by the coding sequence(CDS) (SEQ ID NO: 14) or nucleotide sequence (SEQ ID NO: 13) at ricegene locus LOC_Os06g50890.1. This polypeptide is annotated as “RNArecognition motif containing protein, expressed” in TIGR and “putativetransformer-SR ribonucleoprotein” in NCBI, however does not have anyprior assigned function.

The terms “monocot” and “monocotyledonous plant” are usedinterchangeably herein. A monocot of the current disclosure includes theGramineae.

The terms “dicot” and “dicotyledonous plant” are used interchangeablyherein. A dicot of the current disclosure includes the followingfamilies: Brassicaceae, Leguminosae, and Solanaceae.

The terms “full complement” and “full-length complement” are usedinterchangeably herein, and refer to a complement of a given nucleotidesequence, wherein the complement and the nucleotide sequence consist ofthe same number of nucleotides and are 100% complementary.

An “Expressed Sequence Tag” (“EST”) is a DNA sequence derived from acDNA library and therefore is a sequence which has been transcribed. AnEST is typically obtained by a single sequencing pass of a cDNA insert.The sequence of an entire cDNA insert is termed the “Full-InsertSequence” (“FIS”). A “Contig” sequence is a sequence assembled from twoor more sequences that can be selected from, but not limited to, thegroup consisting of an EST, FIS and PCR sequence. A sequence encoding anentire or functional protein is termed a “Complete Gene Sequence”(“CGS”) and can be derived from an FIS or a contig.

The term “trait” refers to a physiological, morphological, biochemical,or physical characteristics of a plant or particular plant material orcell. In some instances, this characteristic is visible to the humaneye, such as seed or plant size, or can be measured by biochemicaltechniques, such as detecting the protein, starch, or oil content ofseed or leaves, or by observation of a metabolic or physiologicalprocess, e.g. by measuring tolerance to water deprivation or particularsalt or sugar or nitrogen concentrations, or by the observation of theexpression level of a gene or genes, or by agricultural observationssuch as osmotic stress tolerance or yield.

“Agronomic characteristics” is a measurable parameter including but notlimited to, greenness, yield, growth rate, biomass, fresh weight, dryweight at maturation, fruit yield, seed yield, total plant nitrogencontent, fruit nitrogen content, seed nitrogen content, nitrogen contentin vegetative tissue, whole plant amino acid content, vegetative tissuefree amino acid content, fruit free amino acid content, seed free aminoacid content, total plant protein content, fruit protein content, seedprotein content, protein content in a vegetative tissue, droughttolerance, nitrogen uptake, resistance to root lodging, harvest index,stalk lodging, plant height, ear height, and ear length, early seedlingvigor, and seedling emergence under low temperature stress.

“Harvest index” refers to the grain weight divided by the total plantweight.

Increased biomass can be measured, for example, as an increase in plantheight, plant total leaf area, plant fresh weight, plant dry weight orplant grain yield, as compared with control plants.

The ability to increase the biomass or size of a plant would haveseveral important commercial applications. Crop cultivars may bedeveloped to produce higher yield of the vegetative portion of theplant, to be used in food, feed, fiber, and/or biofuel.

Increased leaf size may be of particular interest. Increased leafbiomass can be used to increase production of plant-derivedpharmaceutical or industrial products.

Increased tiller number may be of particular interest and can be used toincrease yield. An increase in total plant photosynthesis is typicallyachieved by increasing leaf area of the plant. Additional photosyntheticcapacity may be used to increase the yield derived from particular planttissue, including the leaves, roots, fruits or seed, or permit thegrowth of a plant under decreased light intensity or under high lightintensity.

Modification of the biomass of another tissue, such as root tissue, maybe useful to improve a plant's ability to grow under harsh environmentalconditions, including nutrient deprivation and/or water deprivation,because larger roots may better reach or take up nutrients and/or water.

“Environmental conditions” refer to conditions under which the plant isgrown, such as the availability of water, availability of nutrients (forexample nitrogen), or the presence of insects or disease.

“Nitrogen limiting conditions” refers to conditions where the amount oftotal available nitrogen (e.g., from nitrates, ammonia, or other knownsources of nitrogen) is not sufficient to sustain optimal plant growthand development. One skilled in the art would recognize conditions wheretotal available nitrogen is sufficient to sustain optimal plant growthand development. One skilled in the art would recognize what constitutessufficient amounts of total available nitrogen, and what constitutessoils, media and fertilizer inputs for providing nitrogen to plants.Nitrogen limiting conditions will vary depending upon a number offactors, including but not limited to, the particular plant andenvironmental conditions.

The terms “nitrogen stress tolerance”, “low nitrogen tolerance” and“nitrogen deficiency tolerance” are used interchangeably herein, whichindicate a trait of a plant and refer to the ability of the plant tosurvive under nitrogen limiting conditions or low nitrogen conditions.

“Increased nitrogen stress tolerance” of a polypeptide indicates thatover-expression of the polypeptide in a transgenic plant confersincreased nitrogen stress tolerance of the transgenic plant relative toa reference or control plant.

“Increased nitrogen stress tolerance” of a plant is measured relative toa reference or control plant, reflects ability of the plant to surviveand/or grow better under nitrogen limiting conditions, and means thatthe nitrogen stress tolerance of the plant is increased by any amount ormeasured when compared to the nitrogen stress tolerance of the referenceor control plant.

A “nitrogen stress tolerant plant” is a plant that exhibits nitrogenstress tolerance. A nitrogen stress tolerant plant can be a plant thatexhibits an increase in at least one agronomic characteristic relativeto a control plant under nitrogen limiting conditions.

“NUE” is nitrogen utilization efficiency and refers to a plant's abilityto utilize nitrogen in low or high levels of fertilizer. It reflects theplant's ability to uptake, assimilate, and/or otherwise utilizenitrogen.

Soil plant analyses development (SPAD) value is SPAD reading which ismeasured by SPAD-502 plus (a chlorophyll meter, made by KONICA MINOLTA).The SPAD value is relative content of leaf chlorophyll and an importantindicator of plant health. Many studies indicated that a significant andpositive correlation was observed between leaf nitrogen content and SPADvalue (Swain D. K. and Sandip S. J. (2010) Journal of Agronomy 9(2):38-44), and leaf SPAD value is used as index of nitrogen statusdiagnosis in crops (Cai H.-G. et al. (2010) Acta metallurgica sinica 16(4): 866-873).

The response and tolerance of rice plants to low nutrition stress is anintegrated and comprehensive physiological and biochemical process. Thetolerance of plants will be reflected in different aspect underdifferent plant development phase and different stress conditions. Theenvironment factors such as illumination and temperature are criticalfactors which effect rice growth, and the variation of these environmentfactors will influence the growth and development of rice plants.Researchers demonstrated that low nitrogen treated rice plants displaylow chlorophyll content in leaf, deduced tiller number, or reducedbiomass. In our experiment, the leaf color (which can be indicated bychlorophyll, SPAD value), plant fresh weight, and tiller number aremeasured, and the low nitrogen tolerance plants are selected bycombining the three parameters.

“Chlorate” refers to a chemical compound containing chlorate anion, asalt of chloric acid. It is a nitrate analog which can be uptake byplant with same transport system like nitrate, and then converted bynitrate reductase to chlorite which is toxic and leads to plant damage,withering, and plant death. Potassium chlorate is used in thisdisclosure.

“Chlorate sensitivity” is a trait of plant, reflects the level ofdamage, even death after chlorate uptake, transport or reduction whentreated with chlorate solution, compared to a reference or controlplant.

“Increased Chlorate sensitivity” of a plant is measured relative to areference or control plant, and reflects higher ability of the plant tochlorate or nitrate uptake, transport or reduce than a reference orcontrol plant in chlorate or nitrate solution. In general, chloratesensitivity can be used as a marker of NUE. The more sensitive plantsare to chlorate, the higher the NUE.

“Chlorate sensitive seedlings” are the damaged seedlings with phenotypeof withered leaves in whole and without green leaf, and considered asdead after treated with chlorate solution.

“Drought” refers to a decrease in water availability to a plant that,especially when prolonged or when occurring during critical growthperiods, can cause damage to the plant or prevent its successful growth(e.g., limiting plant growth or seed yield).

“Drought tolerance” reflects a plant's ability to survive under droughtwithout exhibiting substantial physiological or physical deterioration,and/or its ability to recover when water is restored following a periodof drought.

“Drought tolerance activity” of a polypeptide indicates thatover-expression of the polypeptide in a transgenic plant confersincreased drought tolerance of the transgenic plant relative to areference or control plant.

“Increased drought tolerance” of a plant is measured relative to areference or control plant, and reflects ability of the plant to surviveunder drought conditions with less physiological or physicaldeterioration than a reference or control plant grown under similardrought conditions, or ability of the plant to recover moresubstantially and/or more quickly than would a control plant when wateris restored following a period of drought.

“Paraquat” (1,1-dimethyl-4,4-bipyridinium dichloride), is afoliar-applied and non-selective bipyridinium herbicides, and causesphotooxidative stress which further cause damage to plant or prevent itssuccessful growth.

“Paraquat tolerance” is a trait of a plant, reflects the ability tosurvive and/or grow better when treated with Paraquat solution, comparedto a reference or control plant.

“Increased paraquat tolerance” of a plant is measured relative to areference or control plant, and reflects ability of the plant to survivewith less physiological or physical deterioration than a reference orcontrol plant after treated with paraquat solution. In general,tolerance to relative low level of paraquat can be used as a marker ofabiotic stress tolerance, such as drought tolerance.

“Oxidative stress” reflects an imbalance between the systemicmanifestation of reactive oxygen species and a biological system'sability to readily detoxify the reactive intermediates or to repair theresulting damage. Disturbances in the normal redox state of cells cancause toxic effects through the production of peroxides and freeradicals that damage all components of the cell, including proteins,lipids, and DNA.

“Transgenic” refers to any cell, cell line, callus, tissue, plant partor plant, the genome of which has been altered by the presence of aheterologous nucleic acid, such as a recombinant DNA construct,including those initial transgenic events as well as those created bysexual crosses or asexual propagation from the initial transgenic event.The term “transgenic” as used herein does not encompass the alterationof the genome (chromosomal or extra-chromosomal) by conventional plantbreeding methods or by naturally occurring events such as randomcross-fertilization, non-recombinant viral infection, non-recombinantbacterial transformation, non-recombinant transposition, or spontaneousmutation.

A “control” or “control plant” or “control plant cell” provides areference point for measuring changes in phenotype of a subject plant orplant cell which was genetically altered by, such as transformation, andhas been affected as to a gene of interest. A subject plant or plantcell may be descended from a plant or cell so altered and will comprisethe alteration.

A control plant or plant cell may comprise, for example: (a) a wild-typeplant or cell, i.e., of the same genotype as the starting material forthe genetic alteration which resulted in the subject plant or cell; (b)a plant or plant cell of the same genotype as the starting material butwhich has been transformed with a null construct (i.e., with a constructwhich has no known effect on the trait of interest, such as a constructcomprising a marker gene); (c) a plant or plant cell which is anon-transformed segregant among progeny of a subject plant or plantcell; (d) a plant or plant cell genetically identical to the subjectplant or plant cell but which is not exposed to a condition or stimulusthat would induce expression of the gene of interest; or (e) the subjectplant or plant cell itself, under conditions in which the gene ofinterest is not expressed.

In this disclosure, ZH11-TC, line null, and empty vector plants indicatecontrol plants. ZH11-TC represents rice plants generated from tissuecultured Zhonghua 11, line null represents segregated null plants, andempty vector represents plants transformed with empty vector DP0005 orDP0158.

“Genome” as it applies to plant cells encompasses not only chromosomalDNA found within the nucleus, but organelle DNA found within subcellularcomponents (e.g., mitochondrial, plastid) of the cell.

“Plant” includes reference to whole plants, plant organs, plant tissues,seeds and plant cells and progeny of same. Plant cells include, withoutlimitation, cells from seeds, suspension cultures, embryos, meristematicregions, callus tissue, leaves, roots, shoots, gametophytes,sporophytes, pollen, and microspores.

“Progeny” comprises any subsequent generation of a plant.

“Transgenic plant” includes reference to a plant which comprises withinits genome a heterologous polynucleotide. The heterologouspolynucleotide can be stably integrated within the genome such that thepolynucleotide is passed on to successive generations. The heterologouspolynucleotide may be integrated into the genome alone or as part of arecombinant DNA construct. A T₀ plant is directly recovered from thetransformation and regeneration process. Progeny of T₀ plants arereferred to as T₁ (first progeny generation), T₂ (second progenygeneration), etc.

“Heterologous” with respect to sequence means a sequence that originatesfrom a foreign species, or, if from the same species, is substantiallymodified from its native form in composition and/or genomic locus bydeliberate human intervention.

“Polynucleotide”, “nucleic acid sequence”, “nucleotide sequence”, or“nucleic acid fragment” are used interchangeably and is a polymer of RNAor DNA that is single- or double-stranded, optionally containingsynthetic, non-natural or altered nucleotide bases. Nucleotides (usuallyfound in their 5′-monophosphate form) are referred to by their singleletter designation as follows: “A” for adenine or deoxyadenine (for RNAor DNA, respectively), “C” for cytosine or deoxycytosine, “G” forguanine or deoxyguanine, “U” for uracil, “T” for thymine ordeoxythymine, “R” for purines (A or G), “Y” for pyrimidines (C or T),“K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for anynucleotide.

“Polypeptide”, “peptide”, “amino acid sequence” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical analogue of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers. The terms “polypeptide”, “peptide”, “amino acid sequence”, and“protein” are also inclusive of modifications including, but not limitedto, glycosylation, lipid attachment, sulfation, gamma-carboxylation ofglutamic acid residues, hydroxylation and ADP-ribosylation.

“Messenger RNA (mRNA)” refers to the RNA that is without introns andthat can be translated into protein by the cell.

“cDNA” refers to a DNA that is complementary to and synthesized from anmRNA template using the enzyme reverse transcriptase. The cDNA can besingle-stranded or converted into the double-stranded form using theKlenow fragment of DNA polymerase I.

“Mature” protein refers to a post-translationally processed polypeptide;i.e., one from which any pre- or pro-peptides present in the primarytranslation product has been removed.

“Precursor” protein refers to the primary product of translation ofmRNA; i.e., with pre- and pro-peptides still present. Pre- andpro-peptides may be and are not limited to intracellular localizationsignals.

“Isolated” refers to materials, such as nucleic acid molecules and/orproteins, which are substantially free or otherwise removed fromcomponents that normally accompany or interact with the materials in anaturally occurring environment. Isolated polynucleotides may bepurified from a host cell in which they naturally occur. Conventionalnucleic acid purification methods known to skilled artisans may be usedto obtain isolated polynucleotides. The term also embraces recombinantpolynucleotides and chemically synthesized polynucleotides.

“Recombinant” refers to an artificial combination of two otherwiseseparated segments of sequence, e.g., by chemical synthesis or by themanipulation of isolated segments of nucleic acids by geneticengineering techniques. “Recombinant” also includes reference to a cellor vector, that has been modified by the introduction of a heterologousnucleic acid or a cell derived from a cell so modified, but does notencompass the alteration of the cell or vector by naturally occurringevents (e.g., spontaneous mutation, naturaltransformation/transduction/transposition) such as those occurringwithout deliberate human intervention.

“Recombinant DNA construct” refers to a combination of nucleic acidfragments that are not normally found together in nature. Accordingly, arecombinant DNA construct may comprise regulatory sequences and codingsequences that are derived from different sources, or regulatorysequences and coding sequences derived from the same source, butarranged in a manner different than that normally found in nature.

The terms “entry clone” and “entry vector” are used interchangeablyherein.

“Regulatory sequences” and “regulatory elements” are usedinterchangeably and refer to nucleotide sequences located upstream (5′non-coding sequences), within, or downstream (3′ non-coding sequences)of a coding sequence, and which influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence. Regulatory sequences may include, but are not limited to,promoters, translation leader sequences, introns, and polyadenylationrecognition sequences.

“Promoter” refers to a nucleic acid fragment capable of controllingtranscription of another nucleic acid fragment.

“Promoter functional in a plant” is a promoter capable of controllingtranscription in plant cells whether or not its origin is from a plantcell.

“Tissue-specific promoter” and “tissue-preferred promoter” are usedinterchangeably and refer to a promoter that is expressed predominantlybut not necessarily exclusively in one tissue or organ, but that mayalso be expressed in one specific cell.

“Developmentally regulated promoter” refers to a promoter whose activityis determined by developmental events.

“Operably linked” refers to the association of nucleic acid fragments ina single fragment so that the function of one is regulated by the other.For example, a promoter is operably linked with a nucleic acid fragmentwhen it is capable of regulating the transcription of that nucleic acidfragment.

“Expression” refers to the production of a functional product. Forexample, expression of a nucleic acid fragment may refer totranscription of the nucleic acid fragment (e.g., transcriptionresulting in mRNA or functional RNA) and/or translation of mRNA into aprecursor or mature protein.

“Phenotype” means the detectable characteristics of a cell or organism.

“Introduced” in the context of inserting a nucleic acid fragment (e.g.,a recombinant DNA construct) into a cell, means “transfection” or“transformation” or “transduction” and includes reference to theincorporation of a nucleic acid fragment into a eukaryotic orprokaryotic cell where the nucleic acid fragment may be incorporatedinto the genome of the cell (e.g., chromosome, plasmid, plastid ormitochondrial DNA), converted into an autonomous replicon, ortransiently expressed (e.g., transfected mRNA).

A “transformed cell” is any cell into which a nucleic acid fragment(e.g., a recombinant DNA construct) has been introduced.

“Transformation” as used herein refers to both stable transformation andtransient transformation.

“Stable transformation” refers to the introduction of a nucleic acidfragment into a genome of a host organism resulting in geneticallystable inheritance. Once stably transformed, the nucleic acid fragmentis stably integrated in the genome of the host organism and anysubsequent generation.

“Transient transformation” refers to the introduction of a nucleic acidfragment into the nucleus, or DNA-containing organelle, of a hostorganism resulting in gene expression without genetically stableinheritance.

“Allele” is one of several alternative forms of a gene occupying a givenlocus on a chromosome. When the alleles present at a given locus on apair of homologous chromosomes in a diploid plant are the same thatplant is homozygous at that locus. If the alleles present at a givenlocus on a pair of homologous chromosomes in a diploid plant differ thatplant is heterozygous at that locus. If a transgene is present on one ofa pair of homologous chromosomes in a diploid plant that plant ishemizygous at that locus.

A “chloroplast transit peptide” is an amino acid sequence which istranslated in conjunction with a protein and directs the protein to thechloroplast or other plastid types present in the cell in which theprotein is made. “Chloroplast transit sequence” refers to a nucleotidesequence that encodes a chloroplast transit peptide. A “signal peptide”is an amino acid sequence which is translated in conjunction with aprotein and directs the protein to the secretory system (Chrispeels(1991) Ann. Rev. Plant Phys. Plant Mol. Biol. 42:21-53). If the proteinis to be directed to a vacuole, a vacuolar targeting signal (supra) canfurther be added, or if to the endoplasmic reticulum, an endoplasmicreticulum retention signal (supra) may be added. If the protein is to bedirected to the nucleus, any signal peptide present should be removedand instead a nuclear localization signal included (Raikhel (1992) PlantPhys. 100:1627-1632). A “mitochondrial signal peptide” is an amino acidsequence which directs a precursor protein into the mitochondria (Zhangand Glaser (2002) Trends Plant Sci 7:14-21).

Sequence alignments and percent identity calculations may be determinedusing a variety of comparison methods designed to detect homologoussequences including, but not limited to, the MEGALIGN® program of theLASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison,Wis.). Unless stated otherwise, multiple alignment of the sequencesprovided herein were performed using the Clustal V method of alignment(Higgins and Sharp, CABIOS. 5:151-153 (1989)) with the defaultparameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parametersfor pairwise alignments and calculation of percent identity of proteinsequences using the Clustal V method are KTUPLE=1, GAP PENALTY=3,WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters areKTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignmentof the sequences, using the Clustal V program, it is possible to obtain“percent identity” and “divergence” values by viewing the “sequencedistances” table on the same program; unless stated otherwise, percentidentities and divergences provided and claimed herein were calculatedin this manner.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989(hereinafter “Sambrook”).

Turning now to the embodiments:

Embodiments include isolated polynucleotides and polypeptides,recombinant DNA constructs useful for conferring improved nitrogen useefficiency and/or enhanced drought tolerance, compositions (such asplants or seeds) comprising these recombinant DNA constructs, andmethods utilizing these recombinant DNA constructs.

Isolated Polynucleotides and Polypeptides

The present disclosure includes the following isolated polynucleotidesand polypeptides:

An isolated polynucleotide comprising: (i) a nucleic acid sequenceencoding a polypeptide having an amino acid sequence of at least 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based onthe Clustal V method of alignment, when compared to SEQ ID NO: 6, 9, 12or 15; or (ii) a full complement of the nucleic acid sequence of (i),wherein the full complement and the nucleic acid sequence of (i) consistof the same number of nucleotides and are 100% complementary. Any of theforegoing isolated polynucleotides may be utilized in any recombinantDNA constructs of the present disclosure. The polypeptide is preferablya DN-PPR1, LRP1, DN-LTP1 or RRM1. Over-expression of these polypeptidepreferably increase plant low nitrogen tolerance activity and/or droughttolerance activity.

An isolated polypeptide having an amino acid sequence of at least 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based onthe Clustal V method of alignment, when compared to SEQ ID NO: 6, 9, 12or 15. The polypeptide is preferably an OsDN-PPR1, OsLRP1, OsDN-LTP1 orOsRRM1 polypeptide.

An isolated polynucleotide comprising (i) a nucleic acid sequence of atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity,based on the Clustal V method of alignment, when compared to SEQ ID NO:4, 5, 7, 8, 10, 11, 13 or 14; or (ii) a full complement of the nucleicacid sequence of (i). Any of the foregoing isolated polynucleotides maybe utilized in any recombinant DNA constructs of the present disclosure.The isolated polynucleotide preferably encodes a DN-PPR1, LRP1, DN-LTP1or RRM1 protein. Over-expression of this polypeptide preferably increaseplant low nitrogen tolerance activity and/or drought tolerance activity.

Recombinant DNA Constructs

In one aspect, the present disclosure includes recombinant DNAconstructs.

In one embodiment, a recombinant DNA construct comprises apolynucleotide operably linked to at least one regulatory sequence(e.g., a promoter functional in a plant), wherein the polynucleotidecomprises (i) a nucleic acid sequence encoding an amino acid sequence ofat least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity, based on the Clustal V method of alignment, when compared toSEQ ID NO: 6, 9, 12 or 15; or (ii) a full complement of the nucleic acidsequence of (i).

In another embodiment, a recombinant DNA construct comprises apolynucleotide operably linked to at least one regulatory sequence(e.g., a promoter functional in a plant), wherein said polynucleotidecomprises (i) a nucleic acid sequence of at least 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% sequence identity, based on the Clustal Vmethod of alignment, when compared to SEQ ID NO: 4, 5, 7, 8, 10, 11, 13or 14; or (ii) a full complement of the nucleic acid sequence of (i).

In another embodiment, a recombinant DNA construct comprises apolynucleotide operably linked to at least one regulatory sequence(e.g., a promoter functional in a plant), wherein said polynucleotideencodes a DN-PPR1, LRP1, DN-LTP1 or RRM1 protein. These polypeptidespreferably have low nitrogen tolerance activity and/or drought toleranceactivity, and may be from, for example, Oryza sativa, Arabidopsisthaliana, Zea mays, Glycine max, Glycine tabacina, Glycine soja orGlycine tomentella.

It is understood, as those skilled in the art will appreciate, that thedisclosure encompasses more than the specific exemplary sequences.Alterations in a nucleic acid fragment which result in the production ofa chemically equivalent amino acid at a given site, but do not affectthe functional properties of the encoded polypeptide, are well known inthe art. For example, a codon for the amino acid alanine, a hydrophobicamino acid, may be substituted by a codon encoding another lesshydrophobic residue, such as glycine, or a more hydrophobic residue,such as valine, leucine, or isoleucine. Similarly, changes which resultin substitution of one negatively charged residue for another, such asaspartic acid for glutamic acid, or one positively charged residue foranother, such as lysine for arginine, can also be expected to produce afunctionally equivalent product. Nucleotide changes which result inalteration of the N-terminal and C-terminal portions of the polypeptidemolecule would also not be expected to alter the activity of thepolypeptide. Each of the proposed modifications is well within theroutine skill in the art, as is determination of retention of biologicalactivity of the encoded products.

“Suppression DNA construct” is a recombinant DNA construct which whentransformed or stably integrated into the genome of the plant, resultsin “silencing” of a target gene in the plant. The target gene may beendogenous or transgenic to the plant. “Silencing”, as used herein withrespect to the target gene, refers generally to the suppression oflevels of mRNA or protein/enzyme expressed by the target gene, and/orthe level of the enzyme activity or protein functionality. The terms“suppression”, “suppressing” and “silencing”, used interchangeablyherein, includes lowering, reducing, declining, decreasing, inhibiting,eliminating or preventing. “Silencing” or “gene silencing” does notspecify mechanism and is inclusive, and not limited to, anti-sense,cosuppression, viral-suppression, hairpin suppression, stem-loopsuppression, RNAi-based approaches, and small RNA-based approaches.

A suppression DNA construct may comprise a region derived from a targetgene of interest and may comprise all or part of the nucleic acidsequence of the sense strand (or antisense strand) of the target gene ofinterest. Depending upon the approach to be utilized, the region may be100% identical or less than 100% identical (e.g., at least 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical) to all or part of the sensestrand (or antisense strand) of the gene of interest.

Suppression DNA constructs are well-known in the art, are readilyconstructed once the target gene of interest is selected, and include,without limitation, cosuppression constructs, antisense constructs,viral-suppression constructs, hairpin suppression constructs, stem-loopsuppression constructs, double-stranded RNA-producing constructs, andmore generally, RNAi (RNA interference) constructs and small RNAconstructs such as siRNA (short interfering RNA) constructs and miRNA(microRNA) constructs.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target gene orgene product. “Antisense RNA” refers to an RNA transcript that iscomplementary to all or part of a target primary transcript or mRNA andthat blocks the expression of a target isolated nucleic acid fragment(U.S. Pat. No. 5,107,065). The complementarity of an antisense RNA maybe with any part of the specific gene transcript, i.e., at the 5′non-coding sequence, 3′ non-coding sequence, introns, or the codingsequence.

“Cosuppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of the target gene or geneproduct. “Sense” RNA refers to RNA transcript that includes the mRNA andcan be translated into protein within a cell or in vitro. Cosuppressionconstructs in plants have been previously designed by focusing onover-expression of a nucleic acid sequence having homology to a nativemRNA, in the sense orientation, which results in the reduction of allRNA having homology to the over-expressed sequence (see Vaucheret etal., Plant J. 16:651-659 (1998); and Gura, Nature 404:804-808 (2000)).

Another variation describes the use of plant viral sequences to directthe suppression of proximal mRNA encoding sequences (PCT Publication No.WO 98/36083 published on Aug. 20, 1998).

RNA interference (RNAi) refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire et al., Nature 391:806 (1998)). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing (PTGS) or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire et al., Trends Genet.15:358 (1999)).

Small RNAs play an important role in controlling gene expression.Regulation of many developmental processes, including flowering, iscontrolled by small RNAs. It is now possible to engineer changes in geneexpression of plant genes by using transgenic constructs which producesmall RNAs in the plant.

Small RNAs appear to function by base-pairing to complementary RNA orDNA target sequences. When bound to RNA, small RNAs trigger either RNAcleavage or translational inhibition of the target sequence. When boundto DNA target sequences, it is thought that small RNAs can mediate DNAmethylation of the target sequence. The consequence of these events,regardless of the specific mechanism, is that gene expression isinhibited.

MicroRNAs (miRNAs) are noncoding RNAs of about 19 to about 24nucleotides (nt) in length that have been identified in both animals andplants (Lagos-Quintana et al., Science 294:853-858 (2001),Lagos-Quintana et al., Curr. Biol. 12:735-739 (2002); Lau et al.,Science 294:858-862 (2001); Lee and Ambros, Science 294:862-864 (2001);Llave et al., Plant Cell 14:1605-1619 (2002); Mourelatos et al., Genes.Dev. 16:720-728 (2002); Park et al., Curr. Biol. 12:1484-1495 (2002);Reinhart et al., Genes. Dev. 16:1616-1626 (2002)). They are processedfrom longer precursor transcripts that range in size from approximately70 to 200 nt, and these precursor transcripts have the ability to formstable hairpin structures.

MicroRNAs (miRNAs) appear to regulate target genes by binding tocomplementary sequences located in the transcripts produced by thesegenes. It seems likely that miRNAs can enter at least two pathways oftarget gene regulation: (1) translational inhibition; and (2) RNAcleavage. MicroRNAs entering the RNA cleavage pathway are analogous tothe 21-25 nt short interfering RNAs (siRNAs) generated during RNAinterference (RNAi) in animals and posttranscriptional gene silencing(PTGS) in plants, and likely are incorporated into an RNA-inducedsilencing complex (RISC) that is similar or identical to that seen forRNAi.

Regulatory Sequences:

A recombinant DNA construct (including a suppression DNA construct) ofthe present disclosure may comprise at least one regulatory sequence.

A regulatory sequence may be a promoter.

A number of promoters can be used in recombinant DNA constructs (andsuppression DNA constructs) of the present disclosure. The promoters canbe selected based on the desired outcome, and may include constitutive,tissue-specific, inducible, or other promoters for expression in thehost organism.

Promoters that cause a gene to be expressed in most cell types at mosttimes are commonly referred to as “constitutive promoters”.

High level, constitutive expression of the candidate gene under controlof the 35S or UBI promoter may (or may not) have pleiotropic effects,although candidate gene efficacy may be estimated when driven by aconstitutive promoter. Use of tissue-specific and/or stress-specificpromoters may eliminate undesirable effects, but retain the ability toenhance nitrogen tolerance. This type of effect has been observed inArabidopsis for drought and cold tolerance (Kasuga et al., NatureBiotechnol. 17:287-91 (1999)).

Suitable constitutive promoters for use in a plant host cell include,for example, the core promoter of the Rsyn7 promoter and otherconstitutive promoters disclosed in WO 99/43838 and U.S. Pat. No.6,072,050; the core CaMV 35S promoter (Odell et al., Nature 313:810-812(1985)); rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)); ALS promoter (U.S. Pat. No. 5,659,026), and thelike. Other constitutive promoters include, for example, those discussedin U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142; and 6,177,611.

In choosing a promoter to use in the methods of the disclosure, it maybe desirable to use a tissue-specific or developmentally regulatedpromoter.

A tissue-specific or developmentally regulated promoter is a DNAsequence which regulates the expression of a DNA sequence selectively inthe cells/tissues of a plant critical to tassel development, seed set,or both, and limits the expression of such a DNA sequence to the periodof tassel development or seed maturation in the plant. Any identifiablepromoter may be used in the methods of the present disclosure whichcauses the desired temporal and spatial expression.

Promoters which are seed or embryo-specific and may be useful in thedisclosure include soybean Kunitz trypsin inhibitor (Kti3, Jofuku andGoldberg, Plant Cell 1:1079-1093 (1989)), patatin (potato tubers)(Rocha-Sosa, M., et al., EMBO J. 8:23-29 (1989)), convicilin, vicilin,and legumin (pea cotyledons) (Rerie, W. G., et al., Mol. Gen. Genet.259:149-157 (1991); Newbigin, E. J., et al., Planta 180:461-470 (1990);Higgins, T. J. V., et al., Plant. Mol. Biol. 11:683-695 (1988)), zein(maize endosperm) (Schemthaner, J. P., et al., EMBO J. 7:1249-1255(1988)), phaseolin (bean cotyledon) (Segupta-Gopalan, C., et al., Proc.Natl. Acad. Sci. U.S.A. 82:3320-3324 (1995)), phytohemagglutinin (beancotyledon) (Voelker, T. et al., EMBO J. 6:3571-3577 (1987)),B-conglycinin and glycinin (soybean cotyledon) (Chen, Z-L, et al., EMBOJ. 7:297-302 (1988)), glutelin (rice endosperm), hordein (barleyendosperm) (Marris, C., et al., Plant Mol. Biol. 10:359-366 (1988)),glutenin and gliadin (wheat endosperm) (Colot, V., et al., EMBO J.6:3559-3564 (1987)), and sporamin (sweet potato tuberous root) (Hattori,T., et al., Plant Mol. Biol. 14:595-604 (1990)). Promoters ofseed-specific genes operably linked to heterologous coding regions inchimeric gene constructions maintain their temporal and spatialexpression pattern in transgenic plants. Such examples includeArabidopsis thaliana 2S seed storage protein gene promoter to expressenkephalin peptides in Arabidopsis and Brassica napus seeds(Vanderkerckhove et al., Bio/Technology 7:L929-932 (1989)), bean lectinand bean beta-phaseolin promoters to express luciferase (Riggs et al.,Plant Sci. 63:47-57 (1989)), and wheat glutenin promoters to expresschloramphenicol acetyl transferase (Colot et al., EMBO J. 6:3559-3564(1987)).

Inducible promoters selectively express an operably linked DNA sequencein response to the presence of an endogenous or exogenous stimulus, forexample by chemical compounds (chemical inducers) or in response toenvironmental, hormonal, chemical, and/or developmental signals.Inducible or regulated promoters include, for example, promotersregulated by light, heat, stress, flooding or drought, phytohormones,wounding, or chemicals such as ethanol, jasmonate, salicylic acid, orsafeners.

Promoters for use in the current disclosure include the following: 1)the stress-inducible RD29A promoter (Kasuga et al., Nature Biotechnol.17:287-91 (1999)); 2) the barley promoter, B22E; expression of B22E isspecific to the pedicel in developing maize kernels (“Primary Structureof a Novel Barley Gene Differentially Expressed in Immature AleuroneLayers”, Klemsdal et al., Mol. Gen. Genet. 228(1/2):9-16 (1991)); and 3)maize promoter, Zag2 (“Identification and molecular characterization ofZAG1, the maize homolog of the Arabidopsis floral homeotic geneAGAMOUS”, Schmidt et al., Plant Cell 5(7):729-737 (1993); “Structuralcharacterization, chromosomal localization and phylogenetic evaluationof two pairs of AGAMOUS-like MADS-box genes from maize”, Theissen etal., Gene 156(2):155-166 (1995); NCBI GenBank Accession No. X80206)).Zag2 transcripts can be detected five days prior to pollination to sevento eight days after pollination (“DAP”), and directs expression in thecarpel of developing female inflorescences and Ciml which is specific tothe nucleus of developing maize kernels. Ciml transcript is detectedfour to five days before pollination to six to eight DAP. Other usefulpromoters include any promoter which can be derived from a gene whoseexpression is maternally associated with developing female florets.

For the expression of a polynucleotide in developing seed tissue,promoters of particular interest include seed-preferred promoters,particularly early kernel/embryo promoters and late kernel/embryopromoters. Kernel development post-pollination is divided intoapproximately three primary phases. The lag phase of kernel growthoccurs from about 0 to 10-12 DAP. During this phase the kernel is notgrowing significantly in mass, but rather important events are beingcarried out that will determine kernel vitality (e.g., number of cellsestablished). The linear grain fill stage begins at about 10-12 DAP andcontinues to about 40 DAP. During this stage of kernel development, thekernel attains almost all of its final mass, and various storageproducts (i.e., starch, protein, oil) are produced. Finally, thematuration phase occurs from about 40 DAP to harvest. During this phaseof kernel development the kernel becomes quiescent and begins to drydown in preparation for a long period of dormancy prior to germination.As defined herein “early kernel/embryo promoters” are promoters thatdrive expression principally in developing seed during the lag phase ofdevelopment (i.e., from about 0 to about 12 DAP). “Late kernel/embryopromoters”, as defined herein, drive expression principally indeveloping seed from about 12 DAP through maturation. There may be someoverlap in the window of expression. The choice of the promoter willdepend on the ABA-associated sequence utilized and the phenotypedesired.

Early kernel/embryo promoters include, for example, Cim1 that is active5 DAP in particular tissues (WO 00/11177), which is herein incorporatedby reference. Other early kernel/embryo promoters include theseed-preferred promoters end1 which is active 7-10 DAP, and end2, whichis active 9-14 DAP in the whole kernel and active 10 DAP in theendosperm and pericarp. (WO 00/12733), herein incorporated by reference.Additional early kernel/embryo promoters that find use in certainmethods of the present disclosure include the seed-preferred promoterltp2 (U.S. Pat. No. 5,525,716); maize Zm40 promoter (U.S. Pat. No.6,403,862); maize nuc1c (U.S. Pat. No. 6,407,315); maize ckx1-2 promoter(U.S. Pat. No. 6,921,815 and US Patent Application Publication Number2006/0037103); maize led promoter (U.S. Pat. No. 7,122,658); maize ESRpromoter (U.S. Pat. No. 7,276,596); maize ZAP promoter (U.S. PatentApplication Publication Numbers 20040025206 and 20070136891); maizepromoter eep1 (U.S. Patent Application Publication Number 20070169226);and maize promoter ADF4 (U.S. Patent Application No. 60/963,878, filed 7Aug. 2007). Additional promoters for regulating the expression of thenucleotide sequences of the present disclosure in plants arestalk-specific promoters. Such stalk-specific promoters include thealfalfa S2A promoter (GenBank Accession No. EF030816; Abrahams et al.,Plant Mol. Biol. 27:513-528 (1995)) and S2B promoter (GenBank AccessionNo. EF030817) and the like, herein incorporated by reference.

Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even comprise synthetic DNA segments.

Promoters for use in the current disclosure may include: RIP2, mLIP15,ZmCOR1, Rab17, CaMV 35S, RD29A, B22E, Zag2, SAM synthetase, ubiquitin,CaMV 19S, nos, Adh, sucrose synthase, R-allele, the vascular tissuepreferred promoters S2A (Genbank accession number EF030816) and S2B(GenBank Accession No. EF030817), and the constitutive promoter GOS2from Zea mays. Other promoters include root preferred promoters, such asthe maize NAS2 promoter, the maize Cyclo promoter (US Publication No.2006/0156439, published Jul. 13, 2006), the maize ROOTMET2 promoter (WO2005/063998, published Jul. 14, 2005), the CR1B10 promoter (WO2006/055487, published May 26, 2006), the CRWAQ81 (WO 2005/035770,published Apr. 21, 2005) and the maize ZRP2.47 promoter (NCBI AccessionNo. U38790; NCBI GI No. 1063664).

Recombinant DNA constructs (and suppression DNA constructs) of thepresent disclosure may also include other regulatory sequencesincluding, but not limited to, translation leader sequences, introns,and polyadenylation recognition sequences. In another embodiment of thepresent disclosure, a recombinant DNA construct of the presentdisclosure further comprises an enhancer or silencer.

An intron sequence can be added to the 5′ untranslated region, theprotein-coding region or the 3′ untranslated region to increase theamount of the mature message that accumulates in the cytosol. Inclusionof a spliceable intron in the transcription unit in both plant andanimal expression constructs has been shown to increase gene expressionat both the mRNA and protein levels up to 1000-fold (Buchman and Berg,Mol. Cell Biol. 8:4395-4405 (1988); Callis et al., Genes Dev.1:1183-1200 (1987)).

Any plant can be selected for the identification of regulatory sequencesand genes to be used in recombinant DNA constructs of the presentdisclosure. Examples of suitable plant targets for the isolation ofgenes and regulatory sequences would include but are not limited toalfalfa, apple, apricot, Arabidopsis, artichoke, arugula, asparagus,avocado, banana, barley, beans, beet, blackberry, blueberry, broccoli,brussels sprouts, cabbage, canola, cantaloupe, carrot, cassava,castorbean, cauliflower, celery, cherry, chicory, cilantro, citrus,clementines, clover, coconut, coffee, corn, cotton, cranberry, cucumber,Douglas fir, eggplant, endive, escarole, eucalyptus, fennel, figs,garlic, gourd, grape, grapefruit, honey dew, jicama, kiwifruit, lettuce,leeks, lemon, lime, Loblolly pine, linseed, maize, mango, melon,mushroom, nectarine, nut, oat, oil palm, oil seed rape, okra, olive,onion, orange, an ornamental plant, palm, papaya, parsley, parsnip, pea,peach, peanut, pear, pepper, persimmon, pine, pineapple, plantain, plum,pomegranate, poplar, potato, pumpkin, quince, radiata pine, radicchio,radish, rapeseed, raspberry, rice, rye, sorghum, Southern pine, soybean,spinach, squash, strawberry, sugarbeet, sugarcane, sunflower, sweetpotato, sweetgum, tangerine, tea, tobacco, tomato, triticale, turf,turnip, a vine, watermelon, wheat, yams, and zucchini.

Compositions

A composition of the present disclosure is a plant comprising in itsgenome any of the recombinant DNA constructs (including any of thesuppression DNA constructs) of the present disclosure (such as any ofthe constructs discussed above). Compositions also include any progenyof the plant, and any seed obtained from the plant or its progeny,wherein the progeny or seed comprises within its genome the recombinantDNA construct (or suppression DNA construct). Progeny includessubsequent generations obtained by self-pollination or out-crossing of aplant. Progeny also includes hybrids and inbreds.

In hybrid seed propagated crops, mature transgenic plants can beself-pollinated to produce a homozygous inbred plant. The inbred plantproduces seed containing the newly introduced recombinant DNA construct(or suppression DNA construct). These seeds can be grown to produceplants that would exhibit an altered agronomic characteristic (e.g., anincreased agronomic characteristic optionally under nitrogen limitingconditions), or used in a breeding program to produce hybrid seed, whichcan be grown to produce plants that would exhibit such an alteredagronomic characteristic. The seeds may be maize seeds, or rice seeds.

The plant may be a monocotyledonous or dicotyledonous plant, forexample, a maize or soybean plant, such as a maize hybrid plant or amaize inbred plant. The plant may also be sunflower, sorghum, canola,wheat, alfalfa, cotton, rice, barley or millet.

The recombinant DNA construct is stably integrated into the genome ofthe plant.

Embodiments include but are not limited to the following:

1. A plant (for example, a rice, maize or soybean plant) comprising inits genome a recombinant DNA construct comprising a polynucleotideoperably linked to at least one regulatory sequence, wherein saidpolynucleotide encodes a polypeptide having an amino acid sequence of atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity,based on the Clustal V method of alignment, when compared to SEQ ID NO:6, 9, 12 or 15; and wherein said plant exhibits increased nitrogenstress tolerance and/or drought tolerance when compared to a controlplant not comprising said recombinant DNA construct. The plant mayfurther exhibit an alteration of at least one agronomic characteristicwhen compared to the control plant.

2. A plant (for example, a rice, maize or soybean plant) comprising inits genome a recombinant DNA construct comprising a polynucleotideoperably linked to at least one regulatory sequence, wherein saidpolynucleotide encodes a DN-PPR1, LRP1, DN-LTP1 or RRM1 polypeptide, andwherein said plant exhibits increased nitrogen stress tolerance and/ordrought tolerance when compared to a control plant not comprising saidrecombinant DNA construct. The plant may further exhibit an alterationof at least one agronomic characteristic when compared to the controlplant. The DN-PPR1, LRP1, DN-LTP1 or RRM1 polypeptide may be fromArabidopsis thaliana, Zea mays, Glycine max, Glycine tabacina, Glycinesoja or Glycine tomentella.

3. A plant (for example, a rice, maize or soybean plant) comprising inits genome a recombinant DNA construct comprising a polynucleotideoperably linked to at least one regulatory element, wherein saidpolynucleotide encodes a polypeptide having an amino acid sequence of atleast 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity,based on the Clustal V method of alignment, when compared to SEQ ID NO:6, 9, 12 or 15, and wherein said plant exhibits an alteration of atleast one agronomic characteristic under nitrogen limiting conditionswhen compared to a control plant not comprising said recombinant DNAconstruct.

4. Any progeny of the above plants in embodiment 1-3, any seeds of theabove plants in embodiment 1-3, any seeds of progeny of the above plantsin embodiment 1-3, and cells from any of the above plants in embodiment1-3 and progeny thereof.

In any of the foregoing embodiment 1-4 or any other embodiments of thepresent disclosure, the recombinant DNA construct may comprises at leasta promoter functional in a plant as a regulatory sequence.

In any of the foregoing embodiment 1-4 or any other embodiments of thepresent disclosure, the alteration of at least one agronomiccharacteristic is either an increase or decrease.

In any of the foregoing embodiment 1-4 or any other embodiments of thepresent disclosure, the at least one agronomic characteristic is may beselected from the group consisting of greenness, yield, growth rate,biomass, fresh weight at maturation, dry weight at maturation, fruityield, seed yield, total plant nitrogen content, fruit nitrogen content,seed nitrogen content, nitrogen content in a vegetative tissue, totalplant free amino acid content, fruit free amino acid content, seed freeamino acid content, free amino acid content in a vegetative tissue,total plant protein content, fruit protein content, seed proteincontent, protein content in a vegetative tissue, drought tolerance,nitrogen uptake, root lodging, harvest index, stalk lodging, plantheight, ear length, early seedling vigor, and seedling emergence underlow temperature stress. For example, the alteration of at least oneagronomic characteristic may be an increase in yield, greenness, plantheight or biomass.

In any of the foregoing embodiment 1-4 or any other embodiments of thepresent disclosure, the plant may exhibit the alteration of at least oneagronomic characteristic when compared, under nitrogen stressconditions, to a control plant not comprising said recombinant DNAconstruct.

In any of the foregoing embodiment 1-4 or any other embodiments of thepresent disclosure, the plant may exhibit the alteration of at least oneagronomic characteristic when compared, under drought stress conditions,to a control plant not comprising said recombinant DNA construct.

One of ordinary skill in the art is familiar with protocols forsimulating nitrogen conditions, whether limiting or non-limiting, andfor evaluating plants that have been subjected to simulated ornaturally-occurring nitrogen conditions, whether limiting ornon-limiting. For example, one can simulate nitrogen conditions bygiving plants less nitrogen than normally required or no nitrogen over aperiod of time, and one can evaluate such plants by looking fordifferences in agronomic characteristics, e.g., changes in physiologicaland/or physical condition, including (but not limited to) vigor, growth,size, or root length, or in particular, leaf color or leaf area size.Other techniques for evaluating such plants include measuringchlorophyll fluorescence, photosynthetic rates, root growth or gasexchange rates.

The examples below describe some representative protocols and techniquesfor simulating nitrogen limiting conditions and/or evaluating plantsunder such conditions, simulating drought conditions and/or evaluatingdrought tolerance; and simulating oxidative stress conditions.

One can also evaluate nitrogen stress tolerance by the ability of aplant to maintain sufficient yield (at least 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or 100% yield) in field testing undersimulated or naturally-occurring low or high nitrogen conditions (e.g.,by measuring for substantially equivalent yield under low or highnitrogen conditions compared to normal nitrogen conditions, or bymeasuring for less yield loss under low or high nitrogen conditionscompared to a control or reference plant).

SPAD value can be measured during low or high nitrogen condition in thefield and greenhouse test by a chlorophyll meter. The SPAD value is aparameter indicating the plant health, and reflects plant nitrogencontent by predicting the chlorophyll content. The plants with higherlow nitrogen tolerance will have higher SPAD value compared to a controlor reference plant.

One can also evaluate drought tolerance by the ability of a plant tomaintain sufficient yield (at least 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% yield) in field testing under simulated ornaturally-occurring drought conditions (e.g., by measuring forsubstantially equivalent yield under drought conditions compared tonon-drought conditions, or by measuring for less yield loss underdrought conditions compared to yield loss exhibited by a control orreference plant).

Parameters such as recovery degree, survival rate, paraquat tolerancerate, gene expression level, water use efficiency, level or activity ofan encoded protein, and others are typically presented with reference toa control cell or control plant. A “control” or “control plant” or“control plant cell” provides a reference point for measuring changes inphenotype of a subject plant or plant cell in which genetic alteration,such as transformation, has been effected as to a gene of interest. Asubject plant or plant cell may be descended from a plant or cell soaltered and will comprise the alteration.

One of ordinary skill in the art would readily recognize a suitablecontrol or reference plant to be utilized when assessing or measuring anagronomic characteristic or phenotype of a transgenic plant in anyembodiment of the present disclosure in which a control is utilized(e.g., compositions or methods as described herein). For example, by wayof non-limiting illustrations:

1. Progeny of a transformed plant which is hemizygous with respect to arecombinant DNA construct (or suppression DNA construct), such that theprogeny are segregating into plants either comprising or not comprisingthe recombinant DNA construct (or suppression DNA construct): theprogeny comprising the recombinant DNA construct (or suppression DNAconstruct) would be typically measured relative to the progeny notcomprising the recombinant DNA construct (or suppression DNA construct)(i.e., the progeny not comprising the recombinant DNA construct (or thesuppression DNA construct) is the control or reference plant).

2. Introgression of a recombinant DNA construct (or suppression DNAconstruct) into an inbred line, such as in maize, or into a variety,such as in soybean: the introgressed line would typically be measuredrelative to the parent inbred or variety line (i.e., the parent inbredor variety line is the control or reference plant).

3. Two hybrid lines, where the first hybrid line is produced from twoparent inbred lines, and the second hybrid line is produced from thesame two parent inbred lines except that one of the parent inbred linescontains a recombinant DNA construct (or suppression DNA construct): thesecond hybrid line would typically be measured relative to the firsthybrid line (i.e., the first hybrid line is the control or referenceplant).

4. A plant comprising a recombinant DNA construct (or suppression DNAconstruct): the plant may be assessed or measured relative to a controlplant not comprising the recombinant DNA construct (or suppression DNAconstruct) but otherwise having a comparable genetic background to theplant (e.g., sharing at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or 100% sequence identity of nuclear genetic material comparedto the plant comprising the recombinant DNA construct (or suppressionDNA construct)). There are many laboratory-based techniques availablefor the analysis, comparison and characterization of plant geneticbackgrounds; among these are Isozyme Electrophoresis, RestrictionFragment Length Polymorphisms (RFLPs), Randomly Amplified PolymorphicDNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNAAmplification Fingerprinting (DAF), Sequence Characterized AmplifiedRegions (SCARs), Amplified Fragment Length Polymorphisms (AFLP®s), andSimple Sequence Repeats (SSRs) which are also referred to asMicrosatellites.

Furthermore, one of ordinary skill in the art would readily recognizethat a suitable control or reference plant to be utilized when assessingor measuring an agronomic characteristic or phenotype of a transgenicplant would not include a plant that had been previously selected, viamutagenesis or transformation, for the desired agronomic characteristicor phenotype.

Methods

Methods include but are not limited to methods for increasing nitrogenstress tolerance in a plant, methods for evaluating nitrogen stresstolerance in a plant, methods for increasing chlorate sensitive in aplant, methods for increasing drought tolerance in a plant, methods forevaluating drought tolerance in a plant, methods for increasing paraquattolerance, methods for altering an agronomic characteristic in a plant,methods for determining an alteration of an agronomic characteristic ina plant, and methods for producing seed. The plant may be amonocotyledonous or dicotyledonous plant, for example, a rice, maize,Arabidopsis, soybean plant. The plant may also be sunflower, sorghum,canola, wheat, alfalfa, cotton, barley or millet. The seed may be arice, maize, Arabidopsis or soybean seed, for example a maize hybridseed or maize inbred seed.

Methods include but are not limited to the following:

A method for transforming a cell comprising transforming a cell with anyof the isolated polynucleotides of the present disclosure. The celltransformed by this method is also included. In particular embodiments,the cell is eukaryotic cell, e.g., a yeast, insect or plant cell, orprokaryotic, e.g., a bacterium.

A method for producing a transgenic plant comprising transforming aplant cell with any of the isolated polynucleotides or recombinant DNAconstructs of the present disclosure and regenerating a transgenic plantfrom the transformed plant cell. The disclosure is also directed to thetransgenic plant produced by this method, and transgenic seed obtainedfrom this transgenic plant.

A method for isolating a polypeptide of the disclosure from a cell orculture medium of the cell, wherein the cell comprises a recombinant DNAconstruct comprising a polynucleotide of the disclosure operably linkedto at least one regulatory sequence, and wherein the transformed hostcell is grown under conditions that are suitable for expression of therecombinant DNA construct.

A method of altering the level of expression of a polypeptide of thedisclosure in a host cell comprising: (a) transforming a host cell witha recombinant DNA construct of the present disclosure; and (b) growingthe transformed host cell under conditions that are suitable forexpression of the recombinant DNA construct wherein expression of therecombinant DNA construct results in production of altered levels of thepolypeptide of the disclosure in the transformed host cell.

A method of increasing nitrogen stress tolerance and/or chloratesensitivity in a plant, comprising: (a) introducing into a regenerableplant cell a recombinant DNA construct comprising a polynucleotideoperably linked to at least one regulatory sequence (for example, apromoter functional in a plant), wherein the polynucleotide encodes apolypeptide having an amino acid sequence of at least 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the ClustalV method of alignment, when compared to SEQ ID NO: 6, 9, 12 or 15; and(b) regenerating a transgenic plant from the regenerable plant cellafter step (a), wherein the transgenic plant comprises in its genome therecombinant DNA construct and exhibits increased nitrogen stresstolerance and/or chlorate sensitivity when compared to a control plantnot comprising the recombinant DNA construct. The method may furthercomprise (c) obtaining a progeny plant derived from the transgenicplant, wherein said progeny plant comprises in its genome therecombinant DNA construct and exhibits increased nitrogen stresstolerance and/or chlorate sensitivity when compared to a control plantnot comprising the recombinant DNA construct.

A method of evaluating nitrogen stress tolerance and/or chloratesensitivity in a plant, comprising (a) introducing into a regenerableplant cell a recombinant DNA construct comprising a polynucleotideoperably linked to at least one regulatory sequence (for example, apromoter functional in a plant), wherein the polynucleotide encodes apolypeptide having an amino acid sequence of at least 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the ClustalV method of alignment, when compared to SEQ ID NO: 6, 9, 12 or 15; (b)regenerating a transgenic plant from the regenerable plant cell afterstep (a), wherein the transgenic plant comprises in its genome therecombinant DNA construct; and (c) evaluating the transgenic plant fornitrogen stress tolerance and/or chlorate sensitivity compared to acontrol plant not comprising the recombinant DNA construct. The methodmay further comprise (d) obtaining a progeny plant derived from thetransgenic plant, wherein the progeny plant comprises in its genome therecombinant DNA construct; and (e) evaluating the progeny plant fornitrogen stress tolerance and/or chlorate sensitivity compared to acontrol plant not comprising the recombinant DNA construct.

A method of increasing drought stress tolerance and/or paraquattolerance in a plant, comprising: (a) introducing into a regenerableplant cell a recombinant DNA construct comprising a polynucleotideoperably linked to at least one regulatory sequence (for example, apromoter functional in a plant), wherein the polynucleotide encodes apolypeptide having an amino acid sequence of at least 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the ClustalV method of alignment, when compared to SEQ ID NO: 12; and (b)regenerating a transgenic plant from the regenerable plant cell afterstep (a), wherein the transgenic plant comprises in its genome therecombinant DNA construct and exhibits increased drought stresstolerance and/or paraquat tolerance when compared to a control plant notcomprising the recombinant DNA construct. The method may furthercomprise (c) obtaining a progeny plant derived from the transgenicplant, wherein said progeny plant comprises in its genome therecombinant DNA construct and exhibits increased drought toleranceand/or paraquat tolerance when compared to a control plant notcomprising the recombinant DNA construct.

A method of evaluating drought stress tolerance and/or paraquattolerance in a plant, comprising (a) introducing into a regenerableplant cell a recombinant DNA construct comprising a polynucleotideoperably linked to at least one regulatory sequence (for example, apromoter functional in a plant), wherein the polynucleotide encodes apolypeptide having an amino acid sequence of at least 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity, based on the ClustalV method of alignment, when compared to SEQ ID NO: 12; (b) regeneratinga transgenic plant from the regenerable plant cell after step (a),wherein the transgenic plant comprises in its genome the recombinant DNAconstruct; and (c) evaluating the transgenic plant for drought stresstolerance and/or paraquat tolerance compared to a control plant notcomprising the recombinant DNA construct. The method may furthercomprise (d) obtaining a progeny plant derived from the transgenicplant, wherein the progeny plant comprises in its genome the recombinantDNA construct; and (e) evaluating the progeny plant for drought stresstolerance and/or paraquat tolerance compared to a control plant notcomprising the recombinant DNA construct.

A method of determining an alteration of an agronomic characteristic ina plant, comprising (a) introducing into a regenerable plant cell arecombinant DNA construct comprising a polynucleotide operably linked toat least one regulatory sequence (for example, a promoter functional ina plant), wherein said polynucleotide encodes a polypeptide having anamino acid sequence of at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% sequence identity, based on the Clustal V method of alignment, whencompared to SEQ ID NO: 6, 9, 12 or 15; (b) regenerating a transgenicplant from the regenerable plant cell after step (a), wherein thetransgenic plant comprises in its genome said recombinant DNA construct;and (c) determining whether the transgenic plant exhibits an alterationof at least one agronomic characteristic when compared, optionally undernitrogen limiting conditions and/or drought stress conditions, to acontrol plant not comprising the recombinant DNA construct. The methodmay further comprise (d) obtaining a progeny plant derived from thetransgenic plant, wherein the progeny plant comprises in its genome therecombinant DNA construct; and (e) determining whether the progeny plantexhibits an alteration of at least one agronomic characteristic whencompared, optionally under nitrogen limiting conditions, to a controlplant not comprising the recombinant DNA construct.

A method of producing seed (for example, seed that can be sold as anitrogen stress tolerant product offering) comprising any of thepreceding methods, and further comprising obtaining seeds from saidprogeny plant, wherein said seeds comprise in their genome saidrecombinant DNA construct.

In any of the preceding methods or any other embodiments of methods ofthe present disclosure, the step of determining an alteration of anagronomic characteristic in a transgenic plant, if applicable, maycomprise determining whether the transgenic plant exhibits an alterationof at least one agronomic characteristic when compared, under varyingenvironmental conditions, to a control plant not comprising therecombinant DNA construct.

In any of the preceding methods or any other embodiments of methods ofthe present disclosure, the step of determining an alteration of anagronomic characteristic in a progeny plant, if applicable, may comprisedetermining whether the progeny plant exhibits an alteration of at leastone agronomic characteristic when compared, under varying environmentalconditions, to a control plant not comprising the recombinant DNAconstruct.

In any of the preceding methods or any other embodiments of methods ofthe present disclosure, in said introducing step said regenerable plantcell may comprises a callus cell, an embryogenic callus cell, a gameticcell, a meristematic cell, or a cell of an immature embryo. Theregenerable plant cells may derive from an inbred maize plant.

In any of the preceding methods or any other embodiments of methods ofthe present disclosure, said regenerating step may comprise: (i)culturing said transformed plant cells in a media comprising anembryogenic promoting hormone until callus organization is observed;(ii) transferring said transformed plant cells of step (i) to a firstmedia which includes a tissue organization promoting hormone; and (iii)subculturing said transformed plant cells after step (ii) onto a secondmedia, to allow for shoot elongation, root development or both.

In any of the preceding methods or any other embodiments of methods ofthe present disclosure, the at least one agronomic characteristic may beselected from the group consisting of greenness, yield, growth rate,biomass, fresh weight at maturation, dry weight at maturation, fruityield, seed yield, total plant nitrogen content, fruit nitrogen content,seed nitrogen content, nitrogen content in a vegetative tissue, wholeplant amino acid content, vegetative tissue free amino acid content,fruit free amino acid content, seed free amino acid content, total plantprotein content, fruit protein content, seed protein content, proteincontent in a vegetative tissue, drought tolerance, nitrogen uptake,resistance to root lodging, harvest index, stalk lodging, plant height,ear height, ear length, early seedling vigor, and seedling emergenceunder low temperature stress. The alteration of at least one agronomiccharacteristic may be an increase in yield, greenness, plant height orbiomass.

In any of the preceding methods or any other embodiments of methods ofthe present disclosure, the plant may exhibit the alteration of at leastone agronomic characteristic when compared, under nitrogen stressconditions and/or drought stress conditions, to a control plant notcomprising said recombinant DNA construct.

In any of the preceding methods or any other embodiments of methods ofthe present disclosure, alternatives exist for introducing into aregenerable plant cell a recombinant DNA construct comprising apolynucleotide operably linked to at least one regulatory sequence. Forexample, one may introduce into a regenerable plant cell a regulatorysequence (such as one or more enhancers, optionally as part of atransposable element), and then screen for an event in which theregulatory sequence is operably linked to an endogenous gene encoding apolypeptide of the instant disclosure.

The introduction of recombinant DNA constructs of the present disclosureinto plants may be carried out by any suitable technique, including butnot limited to direct DNA uptake, chemical treatment, electroporation,microinjection, cell fusion, infection, vector mediated DNA transfer,bombardment, or Agrobacterium mediated transformation. Techniques forplant transformation and regeneration have been described inInternational Patent Publication WO 2009/006276, the contents of whichare herein incorporated by reference.

The development or regeneration of plants containing the foreign,exogenous isolated nucleic acid fragment that encodes a protein ofinterest is well known in the art. The regenerated plants areself-pollinated to provide homozygous transgenic plants. Otherwise,pollen obtained from the regenerated plants is crossed to seed-grownplants of agronomically important lines. Conversely, pollen from plantsof these important lines is used to pollinate regenerated plants. Atransgenic plant of the present disclosure containing a desiredpolypeptide is cultivated using methods well known to one skilled in theart.

In general, methods to modify or alter the host endogenous genomic DNAare available. This includes altering the host native DNA sequence or apre-existing transgenic sequence including regulatory elements, codingand non-coding sequences. These methods are also useful in targetingnucleic acids to pre-engineered target recognition sequences in thegenome. As an example, the genetically modified cell or plant describedherein, is generated using “custom” or engineered endonucleases such asmeganucleases produced to modify plant genomes (see e.g., WO2009/114321; Gao et al. (2010) Plant Journal 1:176-187). Anothersite-directed engineering is through the use of zinc finger domainrecognition coupled with the restriction properties of restrictionenzyme. See e.g., Urnov, et al., (2010) Nat Rev Genet. 11(9):636-46;Shukla, et al., (2009) Nature 459 (7245):437-41. A transcriptionactivator-like (TAL) effector-DNA modifying enzyme (TALE or TALEN) isalso used to engineer changes in plant genome. See e.g., US20110145940,Cermak et al., (2011) Nucleic Acids Res. 39(12) and Boch et al., (2009),Science 326(5959): 1509-12. Site-specific modification of plant genomescan also be performed using the bacterial type II CRISPR (clusteredregularly interspaced short palindromic repeats)/Cas (CRISPR-associated)system. See e.g., Belhaj et al., (2013), Plant Methods 9: 39; TheCRISPR/Cas system allows targeted cleavage of genomic DNA guided by acustomizable small noncoding RNA.

EXAMPLES

The present disclosure is further illustrated in the following examples,in which parts and percentages are by weight and degrees are Celsius,unless otherwise stated. It should be understood that these examples,while indicating embodiments of the disclosure, are given by way ofillustration only. From the above discussion and these examples, oneskilled in the art can ascertain the essential characteristic of thisdisclosure, and without departing from the spirit and scope thereof, canmake various changes and modifications of the disclosure to adapt it tovarious usages and conditions. Furthermore, various modifications of thedisclosure in addition to those shown and described herein will beapparent to those skilled in the art from the foregoing description.Such modifications are also intended to fall within the scope of theappended claims.

Example 1 Cloning and Over-Expression Vector Construction of AbioticStress Tolerance Genes

Based on preliminary screening of rice activation tagging population andthe sequences information of gene ID shown in the Table 2, primers weredesigned for cloning rice genes OsDN-PPR1, OsLRP1, OsDN-LNP1 and OsRRM1.The primers and the expected-lengths of the amplified genes are shown inTable 3.

For OsDN-PPR1, OsLRP1 and OsRRM1, their cDNA were cloned by PCR usingpooled cDNA from leaf, stem and root tissues of Zhonghua 11 plant as thetemplate. For OsDN-LNP1, its gDNAs was cloned, and amplified usinggenomic DNA of Zhonghua 11 as the template. The PCR reaction mixturesand PCR procedures are shown in Table 4 and Table 5.

TABLE 2 Rice gene names, Gene IDs (from TIGR) and Construct IDs Genename Gene LOC ID Construct ID OsDN-PPR1 LOC_Os11g10740 DP0039 OsLRP1LOC_Os11g10720 DP0044 OsDN-LTP1 LOC_Os05g41259 DP0047 OsRRM1LOC_Os06g50890 DP0049

TABLE 3 Primers for cloning rice abiotic stress tolerance genesLength of amplified SEQ fragment Primer Sequence ID NO: Gene name (bp)gc-408 5′-GAGCGAACTGCTTGGTTGGGAATG-3′ 16 OsDN-PPR1 1444 gc-4095′-CCCAAAGCATTCATCTCCTCAAATAACG-3 17 gc-403 5′-CGAATGGCCGGCAATGTCATCC-3′18 OsLRP1 2315 gc-404 5′-AGTACCTATTAATCTGTGGTAGCCTCTC-3′ 19 gc-5715′-CCAGGCTACTACTAGTACTCTACCAAC-3′ 20 OsDN-LTP1  749 gc-5725′-CTACGGAGTATATCATTAGATTCACGCTG-3′ 21 gc-2115′-GAGACCGAGAGAGAGAAGCAGCACC-3′ 22 OsRRM1  949 gc-2145′-GGGAGCAACCTTACCTGTCATAGCC-3′ 23

TABLE 4 PCR reaction mixture for cloning abiotic stress tolerance geneReaction mix 50 μL Template  1 μL TOYOBO KOD-FX (1.0 U/μL)  1 μL 2×PCRbuffer for KOD-FX 25 μL 2 mM dNTPs (0.4 mM each) 10 μL Primer-F/R (10μM)  2 μL each ddH₂O  9 μL

TABLE 5 PCR cycle conditions for abiotic stress tolerance gene 94° C. 3min 98° C. 10 s {close oversize brace} ×30 58° C. 30 s 68° C. (1 Kb/min)1 min 68° C. 5 min

The PCR amplified products were extracted from the agarose gel after theelectrophoresis and purified using a column kit and then ligated with TAcloning vectors. The sequences and orientation in these constructs wereconfirmed by DNA sequencing. Then these genes were cloned into plantbinary construct DP0005 (pCAMBIA1300-AsRed) (SEQ ID NO: 1). Thegenerated over-expression vectors were listed in Table 2. The clonednucleotide sequence in construct of DP0039 and coding sequence ofOsDN-PPR1 are provided as SEQ ID NO: 4 and 5, the encoded amino acidsequence of OsDN-PPR1 is SEQ ID NO: 6; the cloned nucleotide sequence inconstruct of DP0044 and coding sequence of OsLRP1 are provided as SEQ IDNO: 7 and 8, the encoded amino acid sequence of OsLRP1 is SEQ ID NO: 9;the cloned nucleotide sequence in construct of DP0047 and codingsequence of OsDN-LNP1 are provided as SEQ ID NO: 10 and 11, the encodedamino acid sequence of OsDN-LNP1 is SEQ ID NO: 12; and the clonednucleotide sequence in construct of DP0049 and coding sequence of OsRRM1are provided as SEQ ID NO: 13 and 14, the encoded amino acid sequence ofOsRRM1 is SEQ ID NO: 15.

DsRed gene expression cassette (SEQ ID NO: 3 in the sequence list) wastransfer to the plant binary construct DP0005 to generate another emptyvector DP0158.

Example 2 Transformation for Transgenic Rice Lines

In this research, all of the over-expression vectors and empty vector(DP0005 and DP0158) were transformed into the Zhonghua 11 (Oryza sativaL.) by Agrobacteria-mediated method as described by Lin and Zhang((2005) Plant Cell Rep. 23:540-547). Zhonghua 11 was cultivated byinstitute of crop sciences, Chinese Academy of Agricultural Sciences.The first batch of seeds used in this research was provided by BeijingWeiming Kaituo Agriculture Biotech Co., Ltd. Calli induced from embryoswas transformed with Agrobacteria with the vector. The transgenicseedlings (T₀) generated in transformation laboratory were transplantedin the field to get T₁ seeds. The T₁ and T₂ seeds were stored at coldroom (4° C.), and T₂ seeds were used for following trait screening.

T₁ transgenic plants were selected by hygromycin by culturing the riceplants (from 1-2 cm in height) in 50 mg/L hygromycin solution, thesurvived plants (hygromycin-resistant) were planted in field to produceT₂ seeds. Only the hygromycin-resistant T₂ transgenic rice was used intrait screen.

Example 3 Gene Expression Analysis

Transgene expression levels of the genes in the transgenic rice plantswere analyzed. A standard RT-PCR or a real-time PCR procedure, such asthe QuantiTect® Reverse Transcription Kit from Qiagen® and RealTime-PCR(SYBR®Premix Ex Taq™, TaKaRa), was used. EF1α gene was used asan internal control to show that the amplification and loading ofsamples from the transgenic rice and wild-type were similar. Geneexpression was normalized based on the EF1α mRNA levels.

As shown in FIG. 1, the expression level of OsLRP1 gene in DP0044.29rice is set at 1.00, OsLRP1 over-expressed in all the ten lines, and noexpression was detected in empty vector transformed plants (DP0005).

(SEQ ID NO: 24) DP0044-1: 5′-CTCCCATCATTTCTCCGCAACTC-3′ (SEQ ID NO: 25)DP0044-2: 5′-CCAAGAGACCCATCCACAACGTC-3′

As shown in FIG. 2. OsLTP1 over-expressed in all the tested lines, whilethe expression levels of OsLTP1 were very low in the both controls ofZH11-TC and DP0158 seedlings.

(SEQ ID NO: 26) DP0047-F1: 5′-GTGCGCATTAAAGAAATTCA-3′ (SEQ ID NO: 27)DP0047-R1: 5′-TCACGCTGACAACACTTTC-3′

Example 4 Greenhouse NUE Screening of Transgenic Rice Plants

In order to investigate whether the genes could improve low nitrogentolerance or nitrogen use efficiency (NUE) in rice plants, thetransgenic rice plants were screened in greenhouse low nitrogen assays.In the greenhouse, two types of lamps are provided as light source, i.e.sodium lamp and metal halide lamp, the ratio is 1:1. Lamps provide the16 h/8 h period of day/night, and are placed approximately 1.5 m abovethe seedbed. The light intensity 30 cm above the seedbed is measured as10,000-20,000 lx in sunny day, while 6,000-10,000 lx in cloudy day, therelative humidity ranges from 30% to 90%, and the temperature rangesfrom 20 to 35° C.

NUE Screening Method:

Transgenic T₂ seeds were sterilized by 800 ppm carbendazol for 8 h at32° C. and washed 3-5 times, then soaked in water for 16 h at 32° C.,germinated for 18 h at 35-37° C. in an incubator. The germinated seedswere selected and planted in pot filled with vermiculite. Randomizedblock design was used in this trait screen. Every screen unit has 4blocks which include 2 controls (ZH11-TC and empty vector, or line null)and 4 transgenic lines. 8 seedlings of each transgenic line were plantedin 4 pots and located in different positions of the 4 blocks. 9-12transgenic lines of each gene were screened.

After cultured for 7-10 days, water was replaced by modified Hoaglandsolution containing 0.75 mM nitrogen (KNO₃) (Table 6). To make aerobiccondition, the nutrition solution was drained off every Monday,Wednesday, and Friday for 2-3 h, and then new modified Hoaglandcontaining low nitrogen solution was added. After cultured in lownitrogen solution for 35-40 days, tiller (including the stem and alltillers) numbers were counted, SPAD value was measured by a SPAD meter(SPAD 502 Plus, made by KONICA MINOLTA) with three different positionsof the second leaf from the top, and the SPAD value was the average ofthree readings; and, the fresh weight of the seedlings (cutting from thejoint of root and stem) was measured by one percent of the balance.After statistical analysis of these data (tiller number, SPAD value andfresh weight), the positive lines were selected by a cut-off of P<0.05.

TABLE 6 Modified Hoagland's nutrient solution for culturing rice MassMolecular formula concentration (g/L) KH₂PO₄ 34.38 MgSO₄•7H₂O 246.50CaCl₂•2H₂O 146.88 KCl 242.29 KNO₃ 101.00 Na₂SiO₃•9H₂O 142.00 H₃BO₃ 1.85MnCl₂•4H₂O 1.98 ZnSO₄•7H₂O 2.87 CuSO₄•5H₂O 0.25 (NH₄)₆MoO₂₄•2H₂O 0.24EDTA-2Na 7.45 FeSO₄•7H₂O 5.57NUE Screening Results1) Validation Results for OsLRP1(DP0044) Transgenic Rice

For OsLRP1 transgenic rice plants, 12 transgenic lines were tested,their line null was used as controls in the first experiment. As shownin Table 7, five lines had greater average tiller numbers, SPAD valuesand fresh weights than their corresponding controls. Two transgeniclines (DP0044.26 and DP0046.30) showed better for these threeparameters. Ten lines exhibited greater tiller numbers. These resultsdemonstrate that the OsLRP1 transgenic rice plants may have enhanced lownitrogen tolerance or improved NUE.

TABLE 7 Low nitrogen assay of OsLRP1 transgenic rice plants undergreenhouse low nitrogen conditions (1^(st) experiment) Tiller numberSPAD value Fresh weight Average Average Average tiller P SPAD P fresh PLine ID number value P ≤ 0.05 value value P ≤ 0.05 weight value P ≤ 0.05DP0044.01 1.4 0.1036 34.68 0.9082 2.886 0.3677 DP0044.01-Null 1.9 34.393.663 DP0044.03 1.1 0.5950 28.27 0.8332 2.230 0.7339 DP0044.03-Null 1.027.36 2.520 DP0044.05 1.6 0.2275 34.83 0.0046 Y 4.098 0.0815DP0044.05-Null 1.1 28.45 2.483 DP0044.07 1.5 0.1970 34.65 0.2418 3.9540.2267 DP0044.07-Null 1.1 33.09 3.385 DP0044.08 1.4 0.1705 31.80 0.28863.233 0.6395 DP0044.08-Null 1.1 33.70 3.503 DP0044.09 1.3 1.0000 35.710.6419 3.585 0.3097 DP0044.09-Null 1.3 36.71 4.465 DP0044.19 1.1 0.350638.71 0.6400 5.076 0.6136 DP0044.19-Null 1.3 38.26 4.871 DP0044.23 1.01.0000 32.90 0.3230 3.521 0.4135 DP0044.23-Null 1.0 35.66 4.073DP0044.26 1.4 0.0796 36.54 0.0555 5.174 0.0376 Y DP0044.26-Null 1.034.69 3.646 DP0044.29 1.0 1.0000 31.58 0.0119 2.559 0.0137DP0044.29-Null 1.0 35.74 3.866 DP0044.30 1.5 0.0796 36.75 0.0160 Y 4.5660.0122 Y DP0044.30-Null 1.1 30.95 2.664 DP0044.31 1.5 0.1970 36.990.1411 4.624 0.0205 Y DP0044.31-Null 1.1 34.03 3.640

In the second experiment, ten lines were tested, ZH11-TC and DP0158seedlings were used as controls, and randomized block design was used.Twelve rice plants from each transgenic line, ZH11-TC and DP0158 wereplanted in one container, repeated twice. When the rice plants grew to3-leaf stage, Hoagland solution containing 0.75 mM potassium nitrate wasapplied to these plants. After low nitrogen stressed for 34 days, tillernumber, SPAD value and fresh weight were measured. The average tillernumber of all the OsLRP1 transgenic rice was significantly greater thanthat of ZH11-TC (P value=0.0408) and greater than that of DP0158 (Pvalue=0.1009) control; the average SPAD value of OsLRP1 transgenic ricewas greater than that of ZH11-TC and DP0158 controls; and the averagefresh weight of OsLRP1 transgenic rice was greater than that of ZH11-TCand DP0158 at construct level.

As shown in Table 8, nine lines exhibited greater tiller number, sixlines exhibited greater SPAD value than ZH11-TC control. As shown inTable 9, nine lines exhibited greater tiller number, eight linesexhibited greater SPAD value, and ten lines exhibited fresh weight thanDP0158 control. These results demonstrate OsLRP1 transgenic riceobtained enhanced low nitrogen tolerance or improved NUE, andover-expression of OsLRP1 enhances NUE of transgenic plants.

TABLE 8 Low nitrogen assay of OsLRP1 transgenic rice plants undergreenhouse low nitrogen conditions (2^(nd) experiment, ZH11-TC ascontrol) Tiller number SPAD value Fresh weight Average Average Averagetiller P SPAD P fresh P Line ID number value P ≤ 0.05 value value P ≤0.05 weight value P ≤ 0.05 DP0044.01 1.7 0.0952 35.05 0.6052 3.1600.9425 DP0044.05 1.8 0.0190 Y 35.56 0.3455 3.474 0.2687 DP0044.07 1.70.0952 34.62 0.8791 3.313 0.6155 DP0044.08 1.8 0.0381 Y 33.61 0.49313.137 0.8723 DP0044.09 2.1 0.0010 Y 36.27 0.1244 3.533 0.1830 DP0044.191.6 0.1251 35.38 0.4300 3.328 0.5766 DP0044.26 1.4 0.5569 32.53 0.11252.921 0.3308 DP0044.29 1.3 0.9296 34.00 0.7195 3.135 0.8676 DP0044.301.6 0.1620 32.39 0.0877 2.961 0.4117 DP0044.31 2.1 0.0002 Y 35.17 0.53723.451 0.3071 ZH11-TC 1.3 34.43 3.180 DP0044 1.7 0.0408 Y 34.46 0.98053.241 0.7898 (construct)

TABLE 9 Low nitrogen assay of OsLRP1 transgenic rice plants undergreenhouse low nitrogen conditions (2^(nd) experiment, DP0158 ascontrol) Tiller number SPAD value Fresh weight Average Average Averagetiller P SPAD P fresh P Line ID number value P ≤ 0.05 value value P ≤0.05 weight value P ≤ 0.05 DP0044.01 1.7 0.1906 35.05 0.0526 3.1600.1921 DP0044.05 1.8 0.0472 Y 35.56 0.0181 Y 3.474 0.0130 Y DP0044.071.7 0.1906 34.62 0.1157 3.313 0.0603 DP0044.08 1.8 0.0865 33.61 0.46193.137 0.2241 DP0044.09 2.1 0.0034 Y 36.27 0.0031 Y 3.533 0.0068 YDP0044.19 1.6 0.2405 35.38 0.0271 Y 3.328 0.0530 DP0044.26 1.4 0.820032.53 0.8682 2.921 0.6862 DP0044.29 1.3 0.6540 34.00 0.2882 3.135 0.2264DP0044.30 1.6 0.2990 32.39 0.7744 2.961 0.5784 DP0044.31 2.1 0.0008 Y35.17 0.0415 Y 3.451 0.0165 Y DP0158 1.4 32.73 2.814 DP0044 1.7 0.100934.46 0.1030 3.241 0.0653 (construct)2) Validation Results for OsDN-LTP1 (DP0047) Transgenic Rice

For OsDN-LTP1 transgenic rice, 12 transgenic lines were tested andZH11-TC and DP0005 seedlings were used as controls in the firstexperiment. When the seedlings grew to 3-leaf stage, Hoagland solutioncontaining 0.75 mM potassium nitrate was applied to these plants for 37days. The average tiller numbers and the fresh weights were greater thanthat of DP0005 controls. As shown in Table 10, three transgenicseedlings had significantly greater fresh weights and two transgeniclines had significantly greater tiller numbers than DP0005 controls.When compared to ZH11-TC controls, ten transgenic lines showed bettertiller numbers. These results indicate that the OsDN-LTP1 transgenicrice plants had enhanced low nitrogen tolerance or improved NUE.

TABLE 10 Low nitrogen assay of OsDN-LTP1 transgenic rice plants undergreenhouse low nitrogen conditions (1^(st) experiment) Tiller numberSPAD value Fresh weight Average Average Average tiller P SPAD P fresh PLine ID number value P ≤ 0.05 value value P ≤ 0.05 weight value P ≤ 0.05DP0047.01 2.6 0.3172 38.60 0.6139 6.411 0.4936 DP0005 2.0 39.29 5.940DP0047.03 2.6 0.0885 37.59 0.3849 5.519 0.4738 DP0005 1.6 39.00 5.021DP0047.06 3.0 0.0014 Y 39.04 0.1189 6.489 0.0006 Y DP0005 1.3 36.934.165 DP0047.16 2.5 0.0032 Y 36.68 0.9947 5.510 0.1550 DP0005 1.4 36.694.553 DP0047.17 2.1 0.1231 34.58 0.6115 5.540 0.0002 Y DP0005 1.4 35.193.640 DP0047.19 2.0 0.7535 39.73 0.0543 6.754 0.0007 Y DP0005 1.9 36.644.613 DP0047.23 1.5 0.0532 36.31 0.2336 4.643 0.5403 DP0005 1.0 37.854.316 DP0047.25 2.4 0.4384 34.65 0.1783 4.310 0.7660 DP0005 2.0 36.284.108

In the second experiment, ten lines were tested, ZH11-TC and DP0158seedlings were used as controls, and randomized block design was used.Twelve rice plants from each transgenic line, ZH11-TC and DP0158 wereplanted in one container. When the rice plants grew to 3-leaf stage,Hoagland solution containing 0.75 mM potassium nitrate was applied tothese plants. After low nitrogen stressed for 35 days, tiller number,SPAD value and fresh weight were measured. The average tiller number,SPAD value and fresh weight of the OsDN-LTP1 transgenic rice were 1.6,34.23 and 3.484 respectively. The average SPAD value was significantlygreater than ZH11-TC control. All the transgenic lines exhibited greatertiller numbers, SPAD values and fresh weights than either ZH11-TC orDP0158 controls (Table 11 and 12). These results demonstrate OsDN-LTP1transgenic rice obtained enhanced low nitrogen tolerance or improvedNUE, and over-expression of OsDN-LTP1 plays a role in enhancing NUE.

TABLE 11 Low nitrogen assay of OsDN-LTP1 transgenic rice plants undergreenhouse low nitrogen conditions (2^(nd) experiment, ZH11-TC ascontrol) Tiller number SPAD value Fresh weight Average Average Averagetiller P SPAD P fresh P Line ID number value P ≤ 0.05 value value P ≤0.05 weight value P ≤ 0.05 DP0047.01 1.6 0.2301 33.61 0.1104 3.4790.2199 DP0047.05 1.6 0.2301 35.14 0.0049 Y 3.489 0.2090 DP0047.06 1.60.2486 32.72 0.3740 3.477 0.2222 DP0047.12 1.6 0.1880 35.12 0.0051 Y3.502 0.1953 DP0047.15 1.6 0.1880 33.29 0.1804 3.479 0.2202 DP0047.161.6 0.2125 34.11 0.0464 Y 3.479 0.2196 DP0047.17 1.6 0.2682 34.07 0.0494Y 3.479 0.2199 DP0047.19 1.6 0.1880 34.26 0.0342 Y 3.495 0.2023DP0047.23 1.6 0.2041 34.88 0.0091 Y 3.485 0.2128 DP0047.25 1.6 0.248635.09 0.0055 Y 3.475 0.2243 ZH11-TC 1.3 31.60 3.078 DP0047 1.6 0.206834.23 0.0169 Y 3.484 0.2099 (construct)

TABLE 12 Low nitrogen assay of OsDN-LTP1 transgenic rice plants undergreenhouse low nitrogen conditions (2^(nd) experiment, DP0158 ascontrol) Tiller number SPAD value Fresh weight Average Average Averagetiller P SPAD P fresh P Line ID number value P ≤ 0.05 value value P ≤0.05 weight value P ≤ 0.05 DP0047.01 1.6 0.2301 33.61 0.3334 3.4790.1790 DP0047.05 1.6 0.2301 35.14 0.0289 Y 3.489 0.1697 DP0047.06 1.60.2486 32.72 0.7951 3.477 0.1810 DP0047.12 1.6 0.1880 35.12 0.0300 Y3.502 0.1580 DP0047.15 1.6 0.1880 33.29 0.4775 3.479 0.1793 DP0047.161.6 0.2125 34.11 0.1732 3.479 0.1788 DP0047.17 1.6 0.2682 34.07 0.18163.479 0.1790 DP0047.19 1.6 0.1880 34.26 0.1366 3.495 0.1639 DP0047.231.6 0.2041 34.88 0.0477 Y 3.485 0.1729 DP0047.25 1.6 0.2486 35.09 0.0317Y 3.475 0.1828 DP0158 1.3 32.39 3.039 DP0047 1.6 0.2068 34.23 0.09493.484 0.1701 (construct)

In the third experiment, the same ten lines were tested, and theexperiment design and the treatment were same to that in the secondexperiment. After low nitrogen stressed for 35 days, tiller number, SPADvalue and fresh weight were measured. The average tiller number, SPADvalue and fresh weight of the OsDN-LTP1 transgenic rice were more thanthat of ZH11-TC and DP0158 controls at construct level. The SPAD valueof OsDN-LTP1 transgenic rice was significantly greater than both ZH11-TCand DP0158 controls; and the tiller number and fresh weight weresignificantly greater than DP0158 seedlings. As shown in Table 13 and14, all the transgenic lines showed greater tiller number, SPAD valueand fresh weights than either ZH11-TC or DP0158 controls. These resultsdemonstrate OsDN-LTP1 transgenic rice obtained enhanced low nitrogentolerance or improved NUE, and OsDN-LTP1 plays a role in enhancing NUEof transgenic plants.

TABLE 13 Low nitrogen assay of OsDN-LTP1 transgenic rice plants undergreenhouse low nitrogen conditions (3^(rd) experiment, ZH11-TC ascontrol) Tiller number SPAD value Fresh weight Average Average Averagetiller P SPAD P fresh P Line ID number value P ≤ 0.05 value value P ≤0.05 weight value P ≤ 0.05 DP0047.01 1.8 0.5251 33.66 0.0926 3.4150.9186 DP0047.05 1.8 0.4647 34.07 0.0472 Y 3.662 0.5429 DP0047.06 1.80.4647 33.77 0.0779 3.682 0.5062 DP0047.12 2.0 0.2270 33.58 0.1050 3.6180.6305 DP0047.15 2.1 0.0749 33.83 0.0698 3.733 0.4164 DP0047.16 2.10.1349 34.09 0.0454 Y 3.537 0.8037 DP0047.17 1.9 0.3090 33.80 0.07333.860 0.2381 DP0047.19 2.1 0.1349 34.18 0.0385 Y 4.027 0.0967 DP0047.231.9 0.2657 33.88 0.0653 3.551 0.7735 DP0047.25 2.0 0.2270 34.01 0.05213.723 0.4328 ZH11-TC 1.6 31.39 3.451 DP0047 1.9 0.2052 33.89 0.05173.681 0.4545 (construct)

TABLE 14 Low nitrogen assay of OsDN-LTP1 transgenic rice plants undergreenhouse low nitrogen conditions (2^(nd) experiment, DP0158 ascontrol) Tiller number SPAD value Fresh weight Average Average Averagetiller P SPAD fresh P Line ID number value P ≤ 0.05 value P value P ≤0.05 weight value P ≤ 0.05 DP0047.01 1.8 0.0897 33.66 0.0290 Y 3.4150.0287 Y DP0047.05 1.8 0.0730 34.07 0.0129 Y 3.662 0.0038 Y DP0047.061.8 0.0730 33.77 0.0236 Y 3.682 0.0031 Y DP0047.12 2.0 0.0232 Y 33.580.0338 Y 3.618 0.0056 Y DP0047.15 2.1 0.0045 Y 33.83 0.0207 Y 3.7330.0019 Y DP0047.16 2.1 0.0106 Y 34.09 0.0123 Y 3.537 0.0111 Y DP0047.171.9 0.0376 Y 33.80 0.0219 Y 3.860 0.0005 Y DP0047.19 2.1 0.0106 Y 34.180.0102 Y 4.027 0.0001 Y DP0047.23 1.9 0.0297 Y 33.88 0.0191 Y 3.5510.0099 Y DP0047.25 2.0 0.0232 Y 34.01 0.0146 Y 3.723 0.0021 Y DP0158 1.330.72 2.656 DP0047 1.9 0.0145 Y 33.89 0.0134 Y 3.681 0.0009 Y(construct)

Example 5 Laboratory Chlorate Screening of Transgenic Rice Plants

Nitrate is a major source of inorganic nitrogen utilized by higherplants. Chlorate is a nitrate analog which can be uptake, transported bythe same system with nitrogen and reduced to a toxic compound (chlorite)by nitrate reductase (NR) in plants. To further confirm the nitrogen useefficiency, chlorate solution is selected to treat seedlings, andseedlings which are sensitive to chlorate will be considered to havebetter nitrogen use efficiency or low nitrogen tolerance.

Laboratory Chlorate Screening Method:

In this assay, over-expression transgenic rice plants from tentransgenic lines were selected and screened by chlorate solution.ZH11-TC and empty vector (DP0158) transgenic plants were used ascontrols.

T₂ transgenic seeds were sterilized and germinated as description inExample 4, and this assay was performed in culture room kept temperatureat 28-30° C. and humidity around ˜30%. The germinated seeds were placedin a tube with a hole at the bottom, and water cultured at 30° C. for 6days till one-leaf and one-terminal bud stage. Uniform seedlings about5.5 cm in height were selected for chlorate screening. Randomized blockdesign was used in this experiment. There are five blocks in onescreened container. Each transgenic line was placed in one row (12plants/line), and ZH11-TC and DP0158 seedlings were placed in 3 rows(3*12 plants) randomly in one block. Then the seedlings were treatedwith 0.4 mM chlorate in concentration for 3-5 days at 10 h day/14 hnight, the treated seedlings first encountered night and uptake thechlorate solution which was changed in the third day. After treated for5 days, the seedlings were then cultured in 1/10 Hoagland's solution(Table 6) for 4 days. The seedlings with withered leaves and totallywithout green are counted as sensitive; while the seedlings only withnecrosed leaves or stem, or bleached leaves are not considered to besensitive seedlings.

Sensitive rate was used as a parameter to for this screen, which is thepercentage of the number of sensitive plants over the total plantnumber.

The data was analyzed at construct level (all transgenic plants comparedto the control) and transgenic line level (different transgenic linescompared to the control) using a statistic model of “Y˜seg+line(seg)+rep+error”, with random effect: “rep”; Statistic Method: “SAS ProcGlimmix”.

Chlorate Screening Results:

1) Validation Results for OsDN-PPR1 (DP0039) Transgenic Rice

In the first experiment, for OsDN-PPR1 transgenic rice, after treatedwith 0.4 mM chlorate solution for 2 days and cultured in 1/10 Hoaglandsolution for 4 days, 227 of the 600 transgenic seedlings (38%) died,while only 14 of the 180 (8%) DP0158 seedlings died, and 32 of the 180(18%) ZH11-TC seedlings died. The sensitive rate of OsDN-PPR1 transgenicseedlings was significantly (P value=0.0000) higher than that of eitherDP0158 or ZH11-TC controls. These results indicate that the OsDN-PPR1transgenic seedlings had enhanced chlorate sensitive rate compared toboth DP0158 and ZH11-TC seedlings at construct level. Table 15 shows theanalysis at transgenic line level. Eight lines exhibited highersensitive rates than both of ZH11-TC and DP0158. Five lines exhibitedsignificantly higher sensitive rates than ZH11-TC control, and eightlines exhibited significantly higher sensitive rates than DP0158seedlings.

TABLE 15 Chlorate sensitive assay of OsDN-PPR1 transgenic rice seedlingsat transgenic line level (1^(st) experiment) Number of Number of deadtotal Sensitive CK = ZH11-TC CK = DP0158 Line ID seedlings seedlingsrate (%) P value P ≤ 0.05 P value P ≤ 0.05 DP0039.04 9 60 15 0.61960.1089 DP0039.07 16 60 27 0.1401 0.0005 Y DP0039.13 5 60 8 0.0895 0.8903DP0039.14 15 60 25 0.2249 0.0012 Y DP0039.15 20 60 33 0.0144 Y 0.0000 YDP0039.18 39 60 65 0.0000 Y 0.0000 Y DP0039.19 33 60 55 0.0000 Y 0.0000Y DP0039.20 36 60 60 0.0000 Y 0.0000 Y DP0039.22 37 60 62 0.0000 Y0.0000 Y DP0039.25 17 60 28 0.0837 0.0002 Y ZH11-TC 32 180 18 DP0158 14180 8

In the second experiment, the same nine transgenic lines were tested.356 of the 600 (59%) OsDN-PPR1 transgenic rice died after chloratetreatment, while 91 of 180 (51%) DP0158 seedlings died and 65 of 180(36%) ZH11-TC seedlings died. The sensitive rate of OsDN-PPR1 transgenicseedlings was significantly higher than that of both DP0158 and ZH11-TCcontrols. Further analysis at transgenic line level indicated four sametransgenic lines (DP0039.07, DP0039.18, DP0039.19 and DP0039.22) showedhigher sensitive rates than DP0158 seedlings (Table 16) and fourtransgenic lines (DP0039.18, DP0039.19, DP0039.22 and DP0039.25) showedhigher sensitive rates than ZH11-TC seedling in two experiments. Theseresults clearly and consistently demonstrate that OsDN-PPR1 transgenicrice plants exhibited enhanced chlorate sensitive compared to DP0158 andZH11-TC seedlings at construct and transgenic line level at seedlingstages. Over-expression of OsDN-PPR1 under CaMV 35S promoter increasedthe chlorate sensitivity of transgenic plants.

TABLE 16 Chlorate sensitive assay of OsDN-PPR1 transgenic rice seedlingsat transgenic line level (2^(nd) experiment) Number of Number of deadtotal Sensitive CK = ZH11-TC CK = DP0158 Line ID seedlings seedlingsrate (%) P value P ≤ 0.05 P value P ≤ 0.05 DP0039.01 42 60 70 0.0000 Y0.0115 Y DP0039.04 28 60 47 0.1501 0.6017 DP0039.07 39 60 65 0.0003 Y0.0567 DP0039.13 39 60 65 0.0003 Y 0.0567 DP0039.14 22 60 37 0.93770.0667 DP0039.18 43 60 72 0.0000 Y 0.0064 Y DP0039.19 45 60 75 0.0000 Y0.0019 Y DP0039.20 28 60 47 0.1501 0.6017 DP0039.22 39 60 65 0.0003 Y0.0567 DP0039.25 31 60 52 0.0368 Y 0.8768 ZH11-TC 65 180 36 DP0158 91180 512) Validation Results of OsDN-LNP1(DP0047) Transgenic Rice

For OsDN-LNP1 transgenic rice, in the first experiment, 266 of the 576transgenic seedlings (46%) died, whereas 67 of the 192 (35%) ZH11-TCseedlings died, and the sensitive rate of OsDN-LNP1 transgenic seedlingswas significantly (P value=0.0112) higher than that of the ZH11-TCcontrol. The result indicates that the OsDN-LNP1 transgenic seedlingshad enhanced chlorate sensitive rate compared to ZH11-TC seedlings atconstruct level.

Further analysis at transgenic line level indicate that seven of the tentransgenic lines had higher sensitive rates than ZH11-TC seedlings, andthe sensitive rates of four transgenic lines were significantly higherthan ZH11-TC seedlings. These results demonstrate that OsDN-LNP1transgenic rice plants have enhanced chlorate sensitive rates comparedto ZH11-TC seedlings at construct and transgenic line level at seedlingstages. OsDN-LNP1 increased the chlorate sensitivity of transgenicplants.

TABLE 17 Chlorate sensitive assay of OsDN-LNP1 transgenic rice seedlingsat transgenic line level (1^(st) experiment) Number Number of dead oftotal Sensitive Line ID seedlings seedlings rate (%) P value P ≤ 0.05DP0047.01 27 60 45 0.1641 DP0047.03 27 60 45 0.1641 DP0047.05 32 60 530.0141 Y DP0047.06 31 60 52 0.0244 Y DP0047.10 42 60 70 0.0000 YDP0047.15 21 60 35 0.9881 DP0047.16 19 60 32 0.6471 DP0047.17 35 60 580.0023 Y DP0047.19 15 36 42 0.4415 DP0047.25 17 60 28 0.3514 ZH11-TC 67192 35

In the second experiment, 273 of the 600 transgenic seedlings (46%)died, whereas 59 of the 180 (33%) ZH11-TC seedlings died. The sensitiverate of OsDN-LNP1 transgenic seedlings was significantly (Pvalue=0.0052) higher than ZH11-TC control. Analysis at transgenic linelevel indicates that eight of the ten transgenic lines had highersensitive rates than ZH11-TC seedlings, and the sensitive rates of fourtransgenic lines were significantly higher than ZH11-TC seedlings (Table18). These results further demonstrate that OsDN-LNP1 transgenic riceplants have enhanced chlorate sensitive rates compared to ZH11-TCseedlings at construct and transgenic line level at seedling stages.OsDN-LNP1 increased the chlorate sensitivity of transgenic plants.

As elucidated in example 4, over-expression of OsDN-LNP1 gene improvednitrogen use efficiency of the transgenic rice. These cross-validationsfurther confirm the increase low nitrogen tolerance or NUE of OsDN-LNP1transgenic rice.

TABLE 18 Chlorate sensitive assay of OsDN-LNP1 transgenic rice seedlingsat transgenic line level (2^(nd) experiment) Number Number Sensitive ofdead of total rate Line ID seedlings seedlings (%) P value P ≤ 0.05DP0047.01 15 60 25 0.2635 DP0047.03 32 60 53 0.0065 Y DP0047.05 21 60 350.7526 DP0047.15 15 60 25 0.2635 DP0047.16 28 60 47 0.0580 DP0047.17 4260 70 0.0000 Y DP0047.19 26 60 43 0.1443 DP0047.20 34 60 57 0.0019 YDP0047.22 28 60 47 0.0580 DP0047.25 32 60 53 0.0066 Y ZH11-TC 59 180 333) Validation Results of OsRRM1(DP0049) Transgenic Rice

In the first experiment, after chlorate treatment, 259 of the 600 (43%)OsRRM1 transgenic seedlings died, while 39 of the 180 (22%) DP0158seedlings died and 57 of the 180 (32%) ZH11-TC seedlings died. Thesensitive rate of OsRRM1 transgenic seedlings was significantly higherthan that of DP0158 (P value=0.0000) and ZH11-TC (P value=0.0192)controls, indicating the OsRRM1 transgenic seedlings had increasedchlorate sensitivity. Further analysis at transgenic line leveldemonstrates that seven of the ten transgenic lines had higher sensitiverates than DP0158 seedlings and ZH11-TC controls. These resultsdemonstrate that OsRRM1 transgenic rice plants had enhanced chloratesensitivity compared to both ZH11-TC and DP0158 seedlings at constructand transgenic line level at seedling stages. Over-expression of OsRRM1under CaMV 35S increased the chlorate sensitivity of transgenic plants.

TABLE 19 Chlorate sensitive assay of OsRRM1 rice seedlings at transgenicline level (1^(st) experiment) Number of Number of dead total SensitiveCK = ZH11-TC CK = DP0158 Line ID seedlings seedlings rate (%) P value P≤ 0.05 P value P ≤ 0.05 DP0049.12 25 60 42 0.1633 0.0040 Y DP0049.13 1060 17 0.0311 0.4091 DP0049.16 26 60 43 0.1061 0.0021 Y DP0049.17 28 6047 0.0405 Y 0.0005 Y DP0049.19 28 60 47 0.0405 Y 0.0005 Y DP0049.20 3760 62 0.0002 Y 0.0000 Y DP0049.26 37 60 62 0.0002 Y 0.0000 Y DP0049.2739 60 65 0.0000 Y 0.0000 Y DP0049.28 10 60 17 0.0311 0.4091 DP0049.32 1960 32 0.9969 0.1229 DP0158 39 180 22 DP0158 39 180 22

In the second experiment, after chlorate treatment, 476 of the 600 (79%)OsRRM1 transgenic seedlings died, while 83 of the 180 (46%) DP0158seedlings died and 83 of the 180 (46%) ZH11-TC seedlings died. Thesensitive rate of OsRRM1 transgenic seedlings was significantly higherthan DP0158 (P value=0.0000) and ZH11-TC (P value=0.0000) controls.These results indicate that the OsRRM1 transgenic seedlings hadincreased chlorate sensitivity. Analysis at transgenic line leveldemonstrates that all the ten transgenic lines exhibited highersensitive rates than both DP0158 and ZH11-TC controls (Table 20). In thetwo experiments, many lines showed better chlorate sensitivity. Theseresults demonstrate that OsRRM1 transgenic rice plants had enhancedchlorate sensitivity compared to both ZH11-TC and DP0158 seedlings atconstruct and transgenic line level at seedling stages. Over-expressionof OsRRM1 under CaMV 35S increased the chlorate sensitivity oftransgenic plants.

TABLE 20 Chlorate sensitive assay of OsRRM1 rice seedlings at transgenicline level (2^(nd) experiment) Number of Number of dead of totalSensitive CK = ZH11-TC CK = DP0158 Line ID seedlings seedlings rate (%)P value P ≤ 0.05 P value P ≤ 0.05 DP0049.12 52 60 87 0.0000 Y 0.0000 YDP0049.13 34 60 57 0.1628 0.1628 DP0049.16 48 60 80 0.0000 Y 0.0000 YDP0049.17 45 60 75 0.0004 Y 0.0004 Y DP0049.19 41 60 68 0.0046 Y 0.0046Y DP0049.20 53 60 88 0.0000 Y 0.0000 Y DP0049.26 58 60 97 0.0000 Y0.0000 Y DP0049.27 52 60 87 0.0000 Y 0.0000 Y DP0049.28 46 60 77 0.0002Y 0.0002 Y DP0049.32 47 60 78 0.0000 Y 0.0000 Y ZH11-TC 83 180 46 DP015883 180 46

Example 6 Field Low Nitrogen Screens of Mature Plants

Field low nitrogen screens were carried out in Beijing. Two nitrogenlevels: N-0 (using fertilizer without nitrogen) and N-1 (with normalfertilizer at 180 kg Nitrogen/ha) were set in this experiment. Seedgermination and seedling culturing were performed as described inExample 4. The germinated seeds were planted in a seedbed field. At3-leaf stage, the seedlings were transplanted into two testing fields,with 4 replicates and 10 plants per replicate for each transgenic line,(the 4 replicates planted in the same block). The ZH11-TC and DP0158plants were nearby the transgenic lines in the same block, and were usedas controls in the statistical analysis.

The rice plants were managed by normal practice using pesticides, butapplying phosphorous fertilizer and potassium fertilizer for N-0treatment and normal fertilizers for N-1.

The SPAD value of the fully expanded flag leaf and top second leaf weremeasured by SPAD-502 chlorophyll meter at about 10 day after heading.The SPAD value of each transgenic rice plant is the arithmetic mean ofSPAD values from three rice plants in the middle of one rice row.

The plant height which is the length from the rice stem base to the endof panicle or the end of the highest leaf was measured at 20 day afterheading. Six rice plants in the middle of one rice row were measured andthe arithmetic mean of these three values is the plant height of thetransgenic rice plant.

At the end of the season, six representative plants of each transgenicline were harvested from the middle of the row per line. The panicleswhich have five seeds are considered as effective panicles, and theeffective panicle number is the total of the effective panicle perplant. The biomass per plant is the dry weight of the rice plant withoutroot and panicle. The SPAD value, plant height, effective number,biomass and grain weight data was statistically analyzed using mixedlinear model by ASRemI program. Positive transgenic lines are selectedbased on the analysis (P≤0.1).

1) Field NUE Validation Results of OsLRP1 (DP0039) Transgenic Rice

As shown in Table 21, the grain yield of OsLRP1 transgenic rice was31.47 g per plant, was lower than that of ZH11-TC and higher than DP0158control under low nitrogen condition at construct level. The similarresults were obtained for the OsLRP1 transgenic rice under normalnitrogen condition (Table 22). There was no difference between OsLRP1transgenic rice and the controls in grain yield, biomass and effectivepanicle number; however OsLRP1 transgenic rice exhibited higher plantheight than both ZH11-TC and DP0158 controls at low nitrogen conditions.

Table 24 demonstrates that two lines were significantly taller thanZH11-TC control and six transgenic lines were significantly taller thanDP0158 control under low nitrogen condition. Table 25 demonstrates thatone transgenic line was significantly taller than ZH11-TC and fourplants were significantly taller than DP0158 control under normalnitrogen conditions. At the construct level, OsLRP1 transgenic riceplants were taller than both ZH11-TC and DP0158 controls under lownitrogen condition, and were shorter than both ZH11-TC and DP0158controls under normal condition. These results demonstrate that OsLRP1transgenic rice plants exhibited enhanced low nitrogen tolerance and/orNUE under low nitrogen field conditions as reflected by plant height.OsLRP1 gene can be used to improve low nitrogen tolerance and/or NUE.

TABLE 21 Grain yield analysis of OsLRP1 transgenic rice under field lownitrogen condition Number of Number of Yield per CK = ZH11-TC CK =DP0158 Line ID survival plants harvested plants plant (g) P value P ≤0.1 P value P ≤ 0.1 DP0039.04 39 24 33.12 0.809 0.169 DP0039.07 40 2430.57 0.357 0.828 DP0039.13 39 24 32.39 0.928 0.292 DP0039.14 40 2429.87 0.214 0.920 DP0039.18 40 24 34.31 0.433 0.055 Y DP0039.19 40 2431.12 0.505 0.640 DP0039.20 40 24 30.93 0.448 0.703 DP0039.22 40 2429.79 0.201 0.891 DP0039.25 40 24 31.11 0.500 0.645 ZH11-TC 39 23 32.59DP0158 39 22 30.09 DP0039 31.47 0.510 0.420 (construct)

TABLE 22 Grain yield analysis of OsLRP1 transgenic rice under fieldnormal nitrogen condition Number of Number of harvested Yield per CK =ZH11-TC CK = DP0158 Line ID survival plants plants plant (g) P value P ≤0.1 P value P ≤ 0.1 DP0039.04 40 24 45.08 0.773 0.232 DP0039.07 39 2442.63 0.237 0.764 DP0039.13 38 20 46.10 0.932 0.119 DP0039.14 40 2043.48 0.382 0.542 DP0039.18 40 24 45.42 0.870 0.187 DP0039.19 40 2446.92 0.703 0.062 Y DP0039.20 40 24 42.31 0.193 0.853 DP0039.22 29 1844.92 0.746 0.285 DP0039.25 39 24 41.81 0.139 1.000 ZH11-TC 40 24 45.87DP0158 40 23 41.80 DP0039 44.30 0.411 0.193 (construct)

TABLE 23 Biomass analysis of OsLRP1 transgenic rice under low nitrogencondition Number of Number of CK = ZH11-TC K = DP0158 Line ID survivalplants harvested plants Biomass (g) P value P ≤ 0.1 P value P ≤ 0.1DP0039.04 39 24 25.45 0.176 0.258 DP0039.07 40 24 24.09 0.566 0.729DP0039.13 39 24 24.51 0.413 0.554 DP0039.14 40 24 24.30 0.487 0.639DP0039.18 40 24 25.13 0.243 0.344 DP0039.19 40 24 24.37 0.463 0.609DP0039.20 40 24 24.08 0.569 0.732 DP0039.22 40 24 25.88 0.109 0.167DP0039.25 40 24 25.63 0.147 0.220 ZH11-TC 39 23 23.08 DP0158 39 22 23.48DP0039 24.82 0.212 0.332 (construct)

TABLE 24 Plant height analysis of OsLRP1 transgenic rice under lownitrogen condition Number of Number of CK = ZH11-TC CK = DP0158 Line IDsurvival plants measured plants Plant height P value P ≤ 0.1 P value P ≤0.1 DP0039.04 39 24 116.73 0.008 Y 0.000 Y DP0039.07 40 24 113.26 0.8740.035 Y DP0039.13 39 24 112.84 0.883 0.083 Y DP0039.14 40 24 112.540.717 0.129 DP0039.18 40 24 114.36 0.336 0.004 Y DP0039.19 40 24 117.440.001 Y 0.000 Y DP0039.20 40 24 108.44 0.001 0.147 DP0039.22 40 24112.12 0.497 0.213 DP0039.25 40 24 113.65 0.654 0.016 Y ZH11-TC 39 24113.05 DP0158 39 24 110.43 (construct) DP0039 113.49 0.773 0.046 Y

TABLE 25 Plant height analysis of OsLRP1 transgenic rice under normalnitrogen condition Number of Number of CK = ZH11-TC CK = DP0158 Line IDsurvival plants measured plants Plant height P value P ≤ 0.1 P value P ≤0.1 DP0039.04 40 24 129.14 0.643 0.017 Y DP0039.07 39 24 128.43 0.8980.064 Y DP0039.13 38 24 125.52 0.006 0.503 DP0039.14 40 24 118.25 0.0000.000 DP0039.18 40 24 128.36 0.845 0.066 Y DP0039.19 40 24 131.91 0.005Y 0.000 Y DP0039.20 40 24 120.72 0.000 0.000 DP0039.22 29 24 125.360.016 0.501 DP0039.25 39 24 126.08 0.032 0.874 ZH11-TC 40 24 128.58DP0158 40 24 126.27 DP0039 125.98 0.068 0.840 (construct)2) Field NUE Validation Results of OsRRM1 (DP0049) Transgenic Rice

The grain yield, biomass, effective panicle number and plant height ofOsRRM1 transgenic rice plants were measured. Table 25 shows that thegrain yield of the OsRRM1 transgenic rice was lower than that of ZH11-TCand DP0158 controls at construct level under low nitrogen conditions,there was no significant difference between the transgenic rice andcontrols. Table 26 shows the grain yield results under field normalnitrogen conditions. The grain yield of OsRRM1 transgenic rice washigher than that of ZH11-TC and DP0158 controls at construct level,eight lines exhibited higher grain yields than ZH11-TC control, and allthe twelve lines exhibited higher grain yields than DP0158 control.There were no significant difference between the OsRRM1 transgenic riceand the controls for the parameters of biomass, effective panicle numberand plant height. These results demonstrate that OsRRM1 transgenic riceplants obtained higher grain yield under normal nitrogen conditions, andlittle decreased grain yield under low nitrogen conditions.

The SPAD values of the plants under low nitrogen conditions weremeasured. As shown in Table 28, the flag leaf SPAD value of OsRRM1transgenic rice plants was 40.58, and was significantly higher than thatof ZH11-TC and DP0158 plants at construct level. At transgenic linelevel, ten lines exhibited significantly higher flag leaf SPAD valuesthan ZH11-TC control, and eight lines exhibited significantly higherflag leaf SPAD values than that of DP0158 control. As shown in Table 29,the top second leaf SPAD value of OsRRM1 transgenic rice plants was39.57, and was significantly higher than that of ZH11-TC and DP0158plants at construct level. At transgenic line level, eleven linesexhibited significantly higher top second leaf SPAD values than ZH11-TCcontrol, and ten lines exhibited significantly higher top second leafSPAD values than that of DP0158 control. These results demonstrate thatOsRRM1 transgenic rice plants showed better growth status than thecontrols under field low nitrogen conditions, OsRRM1 may plays a role inimproving low nitrogen tolerance and/or NUE.

TABLE 26 Grain yield analysis of OsRRM1 transgenic rice under field lownitrogen condition Number of Number of harvested Yield per plant CK =ZH11-TC CK = DP0158 Line ID survival plants plants (g) P value P ≤ 0.1 Pvalue P ≤ 0.1 DP0049.10 39 19 31.43 0.594 0.535 DP0049.12 40 24 30.030.238 0.979 DP0049.13 39 24 30.18 0.266 0.967 DP0049.16 38 20 28.540.063 0.477 DP0049.17 40 24 29.71 0.185 0.860 DP0049.19 37 24 29.610.170 0.826 DP0049.20 36 19 28.71 0.073 0.524 DP0049.22 39 21 24.690.000 0.013 DP0049.26 39 24 31.45 0.599 0.531 DP0049.27 39 24 31.260.541 0.591 DP0049.28 40 24 29.11 0.109 0.650 DP0049.32 40 24 33.010.846 0.177 ZH11-TC 40 24 32.59 DP0158 40 24 30.09 DP0049 29.81 0.0830.862 (construct)

TABLE 27 Grain yield analysis of OsRRM1 transgenic rice under fieldnormal nitrogen condition Number of Number of Yield per CK = ZH11-TC CK= DP0158 Line ID survival plants harvested plants plant (g) P value P ≤0.1 P value P ≤ 0.1 DP0049.10 33 9 50.48 0.140 0.006 Y DP0049.12 40 2444.90 0.723 0.253 DP0049.13 38 20 48.93 0.260 0.009 Y DP0049.16 38 2244.73 0.675 0.279 DP0049.17 38 22 46.26 0.886 0.100 DP0049.19 36 2046.73 0.751 0.070 Y DP0049.20 38 22 47.98 0.436 0.022 Y DP0049.22 31 1742.49 0.280 0.825 DP0049.26 40 23 47.53 0.541 0.035 Y DP0049.27 40 2449.20 0.219 0.006 Y DP0049.28 36 23 46.47 0.823 0.084 Y DP0049.32 39 2044.34 0.598 0.382 ZH11-TC 40 24 45.87 DP0158 40 24 41.80 DP0049 46.670.649 0.006 Y (construct)

TABLE 28 Flag leaf SPAD value analysis of OsRRM1 transgenic rice underfield low nitrogen condition Number of Number of CK = ZH11-TC CK =DP0158 Line ID survival plants measured plants SPAD value P value P ≤0.1 P value P ≤ 0.1 DP0049.10 39 12 40.51 0.028 Y 0.069 Y DP0049.12 4012 39.95 0.160 0.299 DP0049.13 39 12 40.87 0.007 Y 0.020 Y DP0049.16 3812 41.14 0.002 Y 0.006 Y DP0049.17 40 12 40.26 0.062 Y 0.138 DP0049.1937 12 40.26 0.062 Y 0.134 DP0049.20 36 12 40.65 0.017 Y 0.044 YDP0049.22 39 12 39.54 0.394 0.631 DP0049.26 39 12 40.97 0.004 Y 0.012 YDP0049.27 39 12 41.35 0.001 Y 0.002 Y DP0049.28 40 12 40.47 0.031 Y0.076 Y DP0049.32 40 12 40.97 0.005 Y 0.013 Y ZH11-TC 40 12 38.95 DP015840 12 39.21 DP0049 40.58 0.002 Y 0.010 Y (construct)

TABLE 29 Top second leaf SPAD value analysis of OsRRM1 transgenic riceunder field low nitrogen condition Number of Number of CK = ZH11-TC CK =DP0158 Line ID survival plants measured plants SPAD value P value P ≤0.1 P value P ≤ 0.1 DP0049.10 39 12 39.78 0.011 Y 0.022 Y DP0049.12 4012 39.26 0.054 Y 0.093 Y DP0049.13 39 12 40.02 0.005 Y 0.011 Y DP0049.1638 12 40.14 0.003 Y 0.006 Y DP0049.17 40 12 39.29 0.045 Y 0.082 YDP0049.19 37 12 39.55 0.022 Y 0.040 Y DP0049.20 36 12 39.82 0.010 Y0.020 Y DP0049.22 39 12 38.41 0.332 0.470 DP0049.26 39 12 39.54 0.022 Y0.041 Y DP0049.27 39 12 40.35 0.001 Y 0.003 Y DP0049.28 40 12 39.200.060 Y 0.104 DP0049.32 40 12 39.44 0.033 Y 0.058 Y ZH11-TC 40 12 37.57DP0158 40 12 37.79 DP0049 39.57 0.003 Y 0.008 Y (construct)

Example 7 Laboratory Paraquat Assays of Transgenic Rice Plants

Paraquat (1,1-dimethyl-4,4-bipyridinium dichloride), is a foliar-appliedand non-selective bipyridinium herbicide, and it is one of the mostwidely used herbicides in the world, controlling weeds in a huge varietyof crops like corn, rice, soybean etc. In plant cells, paraquat mainlytargets chloroplasts by accepting electrons from photosystem I and thenreacting with oxygen to produce superoxide and hydrogen peroxide, whichcause photooxidative stress. Drought stress and cold stress usuallyleads to increased reactive oxygen species (ROS) in plants andsometimes, the drought and/or cold tolerance of plant is associated withenhanced antioxidative ability. Paraquat is a potent oxidative stressinducer; it greatly increases the ROS production and inhibits theregeneration of reducing equivalents and compounds necessary for theactivity of the antioxidant system. The ROS generation is enhanced underabiotic stress conditions, and the plant responses range from toleranceto death depending on the stress intensity and its associated-ROSlevels. Relative low level of paraquat can mimic the stress-associatedROS production and used as a stress tolerance marker in plant stressbiology (Hasaneen M. N. A. (2012) Herbicide-Properties, Synthesis andControl of Weeds book). Therefore, the paraquat tolerance of the droughttolerant and cold tolerant transgenic rice plants was tested.

Paraquat Assay Methods:

Ten transgenic lines of each transgenic rice line were tested byparaquat assay. Tissue-cultured Zhonghua 11 plants (ZH11-TC) were usedas controls. T₂ transgenic seeds were sterilized and germinated asdescribed in Example 4, and this assay was carried out in growth roomwith temperature at 28-30° C. and humidity ˜30%. The germinated seedswere placed in a tube with a hole at the bottom, and water cultured at30° C. for 5 days till one-leaf and one-terminal bud stage. Uniformseedlings about 3.5-4 cm in height were selected for paraquat testing.Randomized block design was used in this experiment. There were fiveblocks, each of which has 16×12 holes. Each transgenic line was placedin one row (12 plants/line), and ZH11-TC and DP0158 seedlings wereplaced in 3 rows (3×12 plants) randomly in one block. Then the seedlingswere treated with 0.8 μM paraquat solution for 7 days at 10 h day/14 hnight, and the treated seedlings first encountered dark and took up theparaquat solution which was changed every two days. After treated for 7days, the green seedlings were counted. Those seedlings that maintaingreen in whole without damage were considered as paraquat tolerantseedling; those with bleached leaves or stem were not considered asparaquat tolerant seedling.

Tolerance rate was used as a parameter for this trait screen, which isthe percentage of plants which kept green and showed tolerant phenotypeover the total plant number.

The data was analyzed at construct level (all transgenic plants comparedwith the control) and transgenic line level (different transgenic linescompared with the control) using a statistic model of “Y˜seg+line(seg)+rep+error”, random effect of “rep”, Statistic Method of “SAS ProcGlimmix”.

Paraquat Assay Results:

1) Paraquat Validation Results of OsDN-LNP1(DP0047) Transgenic Rice

In the first experiment, after paraquat solution treated, 259 of the 600OsDN-LNP1 transgenic seedlings (43%) kept green and showed tolerantphenotype, while 34 of the 180 (19%) seedlings from ZH11-TC showedtolerant phenotype. The tolerance rate of all screened OsDN-LNP1transgenic seedlings was significantly greater than ZH11-TC (Pvalue=0.0000) control. These results indicate that the OsDN-LNP1transgenic seedlings exhibited enhanced paraquat tolerance compared toZH11-TC control at construct level.

Analysis at transgenic line level indicates that all transgenic linesexhibited greater tolerance rates compared with ZH11-TC control, andnine lines exhibited significantly greater tolerance rates (Table 30).These results demonstrate that OsDN-LNP1 transgenic rice plants hadenhanced paraquat tolerance at construct and transgenic line level atseedling stages. OsDN-LNP1 functions in enhancing paraquat tolerance orantioxidative ability of transgenic plants.

TABLE 30 Paraquat tolerance assay of OsDN-LNP1 transgenic rice plants attransgenic line level (1^(st) experiment) Number of Number of Tolerancetolerant total rate Line ID seedlings seedlings (%) P value P ≤ 0.05DP0047.01 21 60 35 0.0138 Y DP0047.03 15 60 25 0.3145 DP0047.05 31 60 520.0000 Y DP0047.06 28 60 47 0.0001 Y DP0047.12 32 60 53 0.0000 YDP0047.15 26 60 43 0.0005 Y DP0047.17 27 60 45 0.0002 Y DP0047.19 26 6043 0.0005 Y DP0047.23 23 60 38 0.0038 Y DP0047.25 30 60 50 0.0000 YZH11-TC 34 180 19

In the second experiment, the same ten lines were tested. After paraquatsolution treated, 381 of the 600 (64%) OsDN-LNP1 transgenic rice keptgreen and showed tolerant phenotype, whereas 100 of the 180 (56%)ZH11-TC seedlings showed tolerance phenotype. The tolerance rate ofOsDN-LNP1 transgenic rice was significantly greater than ZH11-TC (Pvalue=0.0209) seedlings.

Analysis at transgenic line level was shown in Table 31. Six linesexhibited greater tolerance rates than ZH11-TC control, and four linesshowed significantly greater tolerance rates than ZH11-TC seedlings. Inthe two experiments, many lines exhibited better paraquat tolerance.These results clearly demonstrate that OsDN-LNP1 had enhanced paraquattolerance or antioxidative ability of transgenic plants.

TABLE 31 Paraquat tolerance assay of OsDN-LNP1 transgenic rice plants attransgenic line level (2^(nd) experiment) Number of Number of Tolerancetolerant total rate Line ID seedlings seedlings (%) P value P ≤ 0.05DP0047.01 43 60 72 0.0331 Y DP0047.03 32 60 53 0.7658 DP0047.05 28 60 470.2379 DP0047.06 35 60 58 0.7085 DP0047.12 44 60 73 0.0193 Y DP0047.1528 60 47 0.2379 DP0047.17 50 60 83 0.0005 Y DP0047.19 36 60 60 0.5499DP0047.23 31 60 52 0.6027 DP0047.25 54 60 90 0.0000 Y ZH11-TC 100 180 56

Example 8 Field Drought Assay of Mature Transgenic Rice Plants

Flowering stage drought stress is an important problem in agriculturepractice. The transgenic rice plants were further tested under fielddrought conditions. For the Field drought assays of mature rice plants,12 transgenic lines of each gene construct were tested. The T₂ seedswere first sterilized as described in Example 4. The germinated seedswere planted in a seedbed field. At 3-leaf stage, the seedlings weretransplanted into the testing field, with 4 replicates and 10 plants perreplicate for each transgenic line, and the 4 replicates were planted inthe same block. ZH11-TC and DP0158 were nearby the transgenic lines inthe same block, and were used as controls in the statistical analysis.

The rice plants were managed by normal practice using pesticides andfertilizers. Watering was stopped at the tillering stage, so as to givedrought stress at flowering stage depending on the weather conditions(temperature and humidity). The soil water content was measured every 4days at about 10 sites per block using TDR30 (Spectrum Technologies,Inc.).

Plant phenotypes were observed and recorded during the experiments. Thephenotypes include heading date, leaf rolling degree, droughtsensitivity and/or drought tolerance. Special attention was paid to leafrolling degree at noontime. At the end of the growing season, sixrepresentative plants of each transgenic line were harvested from themiddle of the row per line, and grain weight per plant was measured. Thegrain weight data were statistically analyzed using mixed linear model.Positive transgenic lines were selected based on the analysis (P≤0.1).

Field Drought Assay Results:

Twelve OsDN-LNP2 transgenic lines were tested in Hainan Province in thefirst experiment, ZH11-TC and DP0158 rice plants planted nearby wereused as control. Watering was stopped from panicle initiation stageII-III to seed maturity to produce heavier drought stress. The soilvolumetric moisture content decreased from 35% to 5% during heading andmaturation stage (FIG. 3). Drought stress appeared after 20 days withoutwater, and the rice leaves curled. During this drought stress, DP0047.19rice plants showed greener leaf color and less leaf curl degree thanZH11-TC and DP0158 controls at vegetative stage. 37 days after stoppingwatering, 50% of transgenic rice and ZH11-TC and DP0158 rice plantsstarted heading, and the leaf roll degree increased. DP0047.12,DP0047.19, DP0047.20, DP0047.25 and DP0047.26 showed better seed settingrates at maturation stage.

At the end of the planting season, six representative plants of eachtransgenic line were harvested from the middle of the row per line, andgrain weight per plant was measured. As shown in Table 32, the grainyield of OsDN-LNP1 transgenic rice was 7.13 g per plant, was more thanthat of DP0158 and less than that of ZH11-TC at construct level; fourlines exhibited higher grain yield per plant than that of ZH11-TC andDP1058 controls. These results demonstrate that OsDN-LNP2 rice plantexhibited better drought tolerance at vegetative stage and better grainyield per plant than control after drought stress, and OsDN-LNP1 play arole in enhancing drought tolerance at vegetative stage and may improvethe grain yield at maturation stage.

TABLE 32 Grain yield analysis of OsDN-LNP1 transgenic rice plants underfield drought conditions Number of Number of survival harvested Yieldper CK = ZH11-TC CK = DP0158 Line ID plants plants plant (g) diff Pvalue P ≤ 0.1 diff P value P ≤ 0.1 DP0047.01 40 24 7.01 −0.72 0.360 0.590.455 DP0047.03 38 24 7.52 −0.21 0.788 1.09 0.147 DP0047.12 40 24 8.520.79 0.311 2.10 0.007 Y DP0047.15 40 24 6.28 −1.45 0.064 −0.15 0.852DP0047.16 39 24 7.19 −0.54 0.476 0.76 0.329 DP0047.17 40 24 5.48 −2.240.004 −0.94 0.230 DP0047.19 40 24 6.55 −1.18 0.121 0.12 0.878 DP0047.2040 24 8.08 0.35 0.654 1.65 0.035 Y DP0047.22 39 24 7.23 −0.50 0.523 0.800.293 DP0047.23 40 24 7.89 0.16 0.840 1.46 0.062 Y DP0047.25 40 24 5.81−1.92 0.013 −0.61 0.417 DP0047.26 38 24 7.98 0.25 0.728 1.56 0.037 YZH11-TC 40 24 7.73 DP0158 39 24 6.43 DP0047 7.13 −0.60 0.396 0.70 0.320(construct)

Example 9 Transformation and Evaluation of Maize with Rice Low NitrogenTolerance Genes

Maize plants can be transformed to over-express Oryza sativa lownitrogen tolerance genes or a corresponding homolog from maize,Arabidopsis, or other species. Expression of the gene in the maizetransformation vector can be under control of a constitutive promotersuch as the maize ubiquitin promoter (Christensen et al. (1989) PlantMol. Biol. 12:619-632 and Christensen et al. (1992) Plant Mol. Biol.18:675-689) or under control of another promoter, such as astress-responsive promoter. The recombinant DNA construct can beintroduced into maize cells by particle bombardment substantially asdescribed in International Patent Publication WO 2009/006276.Alternatively, maize plants can be transformed with the recombinant DNAconstruct by Agrobacterium-mediated transformation substantially asdescribed by Zhao et al. in Meth. Mol. Biol. 318:315-323 (2006) and inZhao et al., Mol. Breed. 8:323-333 (2001) and U.S. Pat. No. 5,981,840issued Nov. 9, 1999. The Agrobacterium-mediated transformation processinvolves bacterium inoculation, co-cultivation, resting, selection andplant regeneration.

Progeny of the regenerated plants, such as T₁ plants, can be subjectedto a low nitrogen stress. Using image analysis, plant area, volume,growth rate and color can be measured at multiple times before andduring low nitrogen stress. Significant delay in leaf area reduction, areduced yellow-color accumulation, and/or an increased growth rateduring low nitrogen stress, relative to a control, will be consideredevidence that the gene functions in maize to enhance NUE.

Example 10 Transformation and Evaluation of Gaspe Flint Derived MaizeLines

As described in Example 7, maize plants can be transformed toover-express the rice low nitrogen tolerance genes, or correspondinghomologs from another species. In certain circumstances, recipient plantcells can be from a uniform maize line which having a short life cycle(“fast cycling”), a reduced size, and high transformation potential, andare disclosed in Tomes et al. U.S. Pat. No. 7,928,287.

The population of transgenic (T₀) plants resulting from the transformedmaize embryos can be grown in a controlled greenhouse environment usinga modified randomized block design to reduce or eliminate environmentalerror. For example, a group of 30 plants, comprising 24 transformedexperimental plants and 6 control plants (collectively, a “replicategroup”), are placed in pots which are arranged in an array (a.k.a. areplicate group or block) on a table located inside a greenhouse. Eachplant, control or experimental, is randomly assigned to a location withthe block which is mapped to a unique, physical greenhouse location aswell as to the replicate group. Multiple replicate groups of 30 plantseach may be grown in the same greenhouse in a single experiment. Thelayout (arrangement) of the replicate groups should be determined tominimize space requirements as well as environmental effects within thegreenhouse. Such a layout may be referred to as a compressed greenhouselayout.

Each plant in the line population is identified and tracked throughoutthe evaluation process, and the data gathered from that plant areautomatically associated with that plant so that the gathered data canbe associated with the transgene carried by the plant. For example, eachplant container can have a machine readable label (such as a UniversalProduct Code (UPC) bar code) which includes information about the plantidentity, which in turn is correlated to a greenhouse location so thatdata obtained from the plant can be automatically associated with thatplant.

Alternatively any efficient, machine readable, plant identificationsystem can be used, such as two-dimensional matrix codes or even radiofrequency identification tags (RFID) in which the data is received andinterpreted by a radio frequency receiver/processor (U.S. Pat. Nos.7,403,855 and 7,702,462).

Each greenhouse plant in the T₀ line population, including any controlplants, is analyzed for agronomic characteristics of interest, and theagronomic data for each plant are recorded or stored in a manner so asto be associated with the identifying data for that plant. Confirmationof a phenotype (gene effect) can be accomplished in the T₁ generationwith a similar experimental design to that described above.

Example 11 Laboratory NUE Screening of Rice Low Nitrogen Tolerance Genesin Arabidopsis

To understand whether rice low nitrogen tolerance genes can improvedicot plants' low nitrogen tolerance, or other traits, rice low nitrogentolerance gene over-expression vectors were transformed into Arabidopsis(Columbia) using floral dip method by Agrobacterium mediatedtransformation procedure and transgenic plants were identified (Clough,S. T. and Bent, A. F. (1998) The Plant Journal 16, 735-743; Zhang, X. etal. (2006) Nature Protocols 1: 641-646).

A 16.8-kb T-DNA based binary vector (SEQ ID NO: 2) which is calledpBC-yellow was used in this experiment. This vector contains the RD29apromoter driving expression of the gene for ZS-Yellow, which confersyellow fluorescence to transformed seed. The OsDN-PPR1 and OsRRM1 genewere cloned as described in Example 1, and constructed in the Gatewayvector. Then using the INVITROGEN™ GATEWAY® technology, an LRRecombination Reaction was performed on the entry clone containing thedirectionally cloned PCR product and the pBC-yellow vector to generateGWD0039 and GWD0049 vectors. In these vectors, OsDN-PPR1 and OsRRM1 genewere driven by constitutive promoter CaMV 35S.

Growth Chamber NUE Screening Method:

The T₁ generation fluorescent seeds were selected, surface sterilizedand stratified in the dark at 4° C. for three days. Then 32 T₁individuals were sown next to 32 empty vector control (pBCyellow-emptyvector) individuals on one low nitrogen media containing 0.5× N-FreeHoagland's, 0.4 mM potassium nitrate, 0.1% sucrose, 1 mM MES and 0.25%Phytagel™ as shown in Table 33. Two repeats were prepared. The plateswere horizontally placed in the growth chamber and cultured for a periodof 10 days at 22° C., 60% relative humidity and a 16 hour day cycle.Seedling status was evaluated by imaging the entire plate from 10-13days after stratifications.

After masking the plate image to remove background color, two differentmeasurements are collected for each individual: total rosette area, andthe percentage of color that falls into a green color bin. Using hue,saturation and intensity data (HSI), the green color bin consists ofhues 50 to 66. Total rosette area is used as a measure of plant biomass,whereas the green color bin was shown by dose-response studies to be anindicator of nitrogen assimilation (patent application US20110209245).

The images were analyzed using Nitrosight software and the number ofPixel (for size of the plants) and the intensity of Bin2 (for greencolor of leaves) for each of the 32/64 transgenic seedlings werecompared with 32/64 seedlings of empty vector control for similarparameters. The green color and better growth of the seedling ascompared to the empty vector control seedling signifies improved NUE.The data was statistically analyzed and a gene was considered as a weakvalidation with a P value less than 10⁻⁴ and a strong validation at 10⁻⁵for Bin2 and Area in replicates and multiple days (Day 10 to Day 13 ofassay). In this experiment the statement regarding a positive responsebeing less than 10⁻³ holds.

TABLE 33 Modified Hoagland's nutrient solution for culturing ArabidopsisMolecular formula Molecular weight Concentration (mM) KNO₃ 101.1 0.4MgSO₄•7H₂O 246.49 1.0 CaCl₂ 110.98 2.5 Na₂HPO₄ 141.96 1.0 K₂SO₄ 174.261.3 Fe-EDTA 367.1  4.6 × 10⁻³ MES 195.2 1.0 H₃BO₃ 61.84 12.5 × 10⁻³MnSO₄•H₂O 169.01  1.0 × 10⁻³ ZnSO₄•7H₂O 287.5  1.0 × 10⁻³ CuSO₄•5H₂O249.71 0.25 × 10⁻³ Na₂MoO₄•2H₂O 241.95 0.25 × 10⁻³Growth Chamber NUE Screening Results:1) As shown in Table 34, the P values of the transgenic Arabidopsis'area were lower than 10⁻³ at 10^(th) day in both repeats, and at 11^(th)day in one repeat after stratifications, and the P value of thetransgenic plants' greenness was lower than 10⁻³ at 12^(th) day. Thegrowth status of the OsDN-PPR1 transgenic Arabidopsis was better thanthe empty vector transformed Arabidopsis. These results indicate theOsDN-PPR1 transgenic Arabidopsis grew better than the control in lownitrogen medium. Over-expression of OsDN-PPR1 under CaMV 35S increasedthe low nitrogen tolerance or NUE of a dicot plants.

Over-expression of OsDN-PPR1 increased low nitrogen tolerance or NUE andalso increased the chlorate sensitivity of transgenic rice as describedin Example 5 and 6. This cross-validation further indicates that amonocot gene (OsDN-PPR1 from rice) can function in a dicot plant(Arabidopsis).

TABLE 34 P values for green leaf area and greenness evaluated for theover-expression OsDN-PPR1 on 4 consecutive days under low nitrogencondition 10^(th) Day 11^(th) Day 12^(th) Day 13^(th) Day Bin2- Area-Bin2- Area- Bin2- Area- Bin2- Area- Plate # p-value p-value p-valuep-value p-value p-value p-value p-value a 8.57E−01 1.49E−03 9.66E−016.61E−04 9.01E−01 1.42E−01 3.88E−02 2.33E−01 b 2.83E−01 4.49E−036.99E−02 1.09E−02 1.28E−03 6.92E−02 9.52E−01 7.33E−022) the P values of OsRRM1-transgenic Arabidopsis area were lower than10⁻³ at 11^(th) day one repeat after stratifications, and the P value ofthe transgenic plants' greenness was lower than 10⁻³ at 10^(th) and11^(th) day in both repeat and was lower than 10⁻³ at 12^(th) and13^(th) in one repeat (Table 35), indicating the OsRRM1 transgenicArabidopsis grew better than the control in low nitrogen medium.Over-expression of OsRRM1 under CaMV 35S increased the low nitrogentolerance or NUE of a dicot plants. Over-expression of OsRRM1 increasedlow nitrogen tolerance or NUE and also increased the chloratesensitivity of transgenic rice as described in Example 5 and 6. Thiscross-validation further indicates that a monocot gene (OsRRM1 fromrice) can function in a dicot plant (Arabidopsis).

TABLE 35 P values for green leaf area and greenness evaluated for theover expression OsRRM1 on 4 consecutive days under low nitrogencondition 10^(th) Day 11^(th) Day 12^(th) Day 13^(th) Day Bin2- Area-Bin2- Area- Bin2- Area- Bin2- Area- Plate # p-value p-value p-valuep-value p-value p-value p-value p-value a 1.46E−07 8.76E−03 2.78E−041.57E−02 4.61E−07 2.72E−02 1.84E−01 3.09E−02 b 2.28E−03 5.25E−022.71E−04 2.48E−02 2.11E−02 1.70E−02 8.53E−03 1.89E−02

What is claimed is:
 1. A method of increasing nitrogen stress tolerancein a plant, comprising: (a) expressing a polynucleotide in a plant cell,wherein the polynucleotide encodes a polypeptide having an amino acidsequence of at least 95% sequence identity when compared to SEQ ID NO:12 and wherein the polynucleotide is under the control of a heterologousregulatory element; and (b) obtaining a plant from the plant cell,wherein said plant exhibits increased nitrogen stress tolerance whencompared to a control plant not comprising the heterologous regulatoryelement.
 2. The method of claim 1, wherein the progeny plant exhibitsincreased yield.
 3. The method of claim 1, wherein the plant is rice ormaize.
 4. The method of claim 1, wherein the heterologous regulatoryelement is a promoter.